key: cord-0034113-iizp0xxm
authors: nan
title: Satellite Symposia and Meet-the-Professor Sessions: Satellite Symposium I
date: 2006-01-20
journal: Ann Hematol
DOI: 10.1007/s00277-005-0079-8
sha: c7bb5f11a999785c8095552a39ae321f2578f730
doc_id: 34113
cord_uid: iizp0xxm

nan

One of the prime objectives of the planned new generation of the AML-CG studies will be the evaluation of dose density of AraC during the induction phase. In the past total dose was increased by implementing high-dose AraC regimens (HAM -6 to 18g/m2 per cycle) in place of standard dose AraC regimens (TAD 1,4g/m2 per cycle) during the induction phase. Thus the TAD-TAD double induction regimen has been progressively replaced by first the TAD-HAM regimen and now (following the last study generation AML-CG 1999) by the HAM-HAM regimen. This increase in total dose has yielded improved long term survival especially in patients with prognostically unfavourable characteristics. However since a) further increases in AraC dose are not feasible and b) leukemic regrowth between treatment cycles is expected to compromise long term results [1, 2] the new study generation will now increase dose density of AraC by reducing the treatment interval between two HAM cycles (from 17 days) to a minimum of 3 days thereby more than doubling dose intensity.

The current pilot study is therefore intended to establish the feasibility of such a dose-dense protocol. In a first step the S-HAM protocol (originally intended for relapsed or refractory AML) was tested in first-line therapy because this regime already features the desired characteristics of a dose-dense double HAM regimen with a between cycle interval of only 3 days [3] . However since in this protocol both the high-dose AraC as well as the mitoxantrone are applied only on 2 days respectively as compared to 3 days as is the case in the conventional HAM regimen only a total dose of 66% is applied during S-HAM induction as compared to the 100% of standard HAM-HAM double induction. After establishing the feasibility of S-HAM (66% dose) in first-line therapy in a second step the total dose of the S-HAM regimen will therefore be increased to equal that of HAM-HAM double induction (S-HAM 100% dose). When feasibility of this dose-dense protocol is established the S-HAM (100% dose) regimen will constitute the new experimental arm for the phase III randomized AML-CG study comparing conventional HAM-HAM double induction as the standard arm to the experimental dose-dense S-HAM (100% dose) regimen. Mathematical modelling derived from the previous AML-CG studies have indicated that such an increase in dose-density is expected to result in a 5 -11% increase in event free survival [4] .

Currently the feasibility of the conventional S-HAM (66% dose) regimen in first line therapy could be demonstrated. As of 09/2005 67 patients have been included into this pilot study of whom 51 were evaluable for response, 50 for critical neutropenia and 51 for grade 3/4 non-hematological toxicity. As shown in table 1 the early death rate (ED) was 6% (3 out of 51) and therefore does not exceed the ED rate observed previously in less dose-dense regimens (TAD-TAD 18%, TAD-HAM 12% 5,6) but rather shows a trend towards a decreased early mortality. In parallel a substantial antileukemic efficacy could be demonstrated with 71% of patients reaching a complete remission (CR) which compares favourably to results of TAD-TAD double induction (65% CR) and HAM-HAM (60% CR) [5, 6] ).

For quantification of hematological toxicities recovery of >500/l neutrophils was measured from the start of S-HAM (66% dose) therapy. As shown in figure 1 a median duration of critical neutropenia of 29 days was observed which is substantially less than the time to recovery from the start of double induction therapy (TAD-TAD 46 days, TAD-HAM 46 days [5, 6] ).

Regarding non-hematological toxicities expected grade IV toxicities were for infections during critical neutropenia 33%). Also grade III to IV toxicities for rises in bilirubin levels were observed in 6% of patients most likely due to the known hepatotoxic effects of high-dose AraC. In 8% of patients grade III and IV cutaneous toxicities were observed. These data are summarized in table 2 below.

In summary in the first step of implementation of a dose-dense regimen in first line AML therapy (S-HAM -66% dose) no excess toxicity or ED rate was observed. Rather critical neutropenia was considerably shorter than in historical controls and the high antileukemic efficacy was demonstrated by a low rate of persistent leukemias. Therefore in the next step the full dose of AraC and mitoxantrone is aimed for (S-HAM100% dose) and the feasibility of this approach will be checked. The next cohort of patients (n=20) will receive the first 5 days of therapy as in the regular HAM regimen but will then after a treatmentfree interval of three days receive the second part of therapy as in the S-HAM66% dose regimen -resulting in a total of 83% of the aimed for dose. Following this cohort of patients -provided no excess mortality as defined is observed -the remaining patients of the study will be treated with aimed for final dose (S-HAM100% dose). Again early stopping rules will apply in all remaining therapy phases indicating that the present trial will be stopped prematurely if the lower boundary of the 95%-confidence interval for the ED rate exceeds 20%. shortens duration of critical neutropenia and prolongs disease-free survival after sequential high-dose cytosine arabinoside and mitoxantrone (S-HAM) salvage therapy for refractory or relapsed acute myeloid leukemia. Ann Hematol. 1998;77:115 -122. Growth factor priming in AML has been investigated by 12 major randomized trials published during the past 10 years [overview 1]. GM-CSF or G-CSF were administered in connection with one [1] [2] [3] , routinely two [4] or occasionally two [5] induction chemotherapy courses, and started 48 hours [5] or 24 hours [2, 4] before chemotherapy, together with the start of chemotherapy [1] or 12 hours thereafter [3] . The first chemotherapy courses consisted of standard dose araC [1] [2] [3] [4] [5] . While the addition of anthracyclines mostly started on day 1 of araC [1] [2] [3] 5] , their addition was delayed until day 6 [4] .

A therapeutic benefit was shown in 3 out of the 12 trials [1, 3, 4] . Thus, in 722 patients of 61 to 80 years with de-novo or secondary AML glycosylated G-CSF during and after chemotherapy was associated with a higher complete remission rate (p=0.009) [1] . In 240 patients of 50 to 75 years with de-novo AML GM-CSF during and after chemotherapy led to improved disease-free survival (p= 0.003) and overall survival (p=0.082) [3] . In 640 patients with denovo or secondary AML 18 to 60 years of age the addition of G-CSF starting 1 day prior to the start of araC and 7 days prior to the first dose of anthracyclin resulted in a superior disease-free survival (p=0.02) and remission duration (p=0.04), and in the subgroup of standard risk in longer disease-free survival (p=0.006) and overall survival (p=0.02) [4] .

In the AMLCG 1999 trial patients 16 to 85 years of age with de-novo or secondary AML received for induction standard dose/high dose or high dose/ high dose araC combinations, standard consolidation, prolonged maintenance or autologous stem cell transplantation. By randomization G-CSF was given with all chemotherapy courses during the first year and started 48 hours before each course. AraC was given alone on the first 2 days of all courses before anthracyclines were added. Confirming a preliminary report [6] a new update shows identical complete remission rate, overall survival, and disease-free survival (figure 1) both in patients <60 years and those 60 years of age and older. It will take another two years of observation to detect possible therapeutic effects of G-CSF priming in particular prognostic subsets of patients with AML.

Morbidity and mortality in patients with hematological malignancies are increased by invasive fungal infections. Since diagnosis of invasive fungal infection is often delayed, antifungal prophylaxis is an attractive approach for patients expecting prolonged neutropenia or allogeneic stem cell transplantation. Antifungal prophylaxis has obviously attracted much interest resulting in more than 50 clinical trials since the late 1970s.

Allogeneic stem cell transplant recipients are at particularly high risk for invasive fungal infections. Other well described risk factors are neutropenia >10 days, corticosteroid therapy, sustained immunosuppression, graft versus host disease, and concomitant viral infections. The enormous study efforts are contrasted by a scarcity of risk stratified evidence based recommendations for clinical decision making. The objective of this review accumulating information on about 11.000 patients is to assess evidence based criteria primarily regarding the efficacy of antifungal prophylaxis in neutropenic cancer patients. The impact of evidence based medicine (EBM) on therapeutic decisions is increasing. Several meta-analyses on prophylaxis of invasive fungal infections are at hand, but do not differentiate between specific patient populations and risk factors [1] [2] [3] . This state of the art article aims at giving the treating physician a tool for the daily bed side decisions on antifungal prophylaxis. The EBM criteria proposed by the Infectious Diseases Society of America (IDSA) are used throughout this document [4] .

The steadily rising incidence of systemic fungal infections compromises therapeutic outcomes in cancer patients and transplant recipients. A causal relationship is seen to intensified chemotherapy regimens, stem cell, bone marrow and solid organ transplantation [5, 6] . Improving the effectiveness of diagnostic procedures including non-cultural methods remains an unresolved task. Complex and costly treatments and moreover a high case fatality rate still dominate the daily practice [7] .

The decision to institute antifungal prophylaxis depends on the goal to be achieved. Reducing the incidence of invasive fungal infection is an essential target, but in the light of fundamental diagnostic uncertainties such an endpoint is hard to reliably evaluate. Despite the advent of new potent antifungal drugs invasive fungal infection still translates into death from fungal infection in a high percentage of patients. Thus, looking for the most relevant target of antifungal prophylaxis leads to pursuing the reduction of the mortality attributable to invasive fungal infection.

One Approach Fits all? The incidence of invasive fungal infection is high in hematological malignancies. However, a wide range of incidence rates of proven and probable invasive aspergillosis has to be dealt with in a context of varying case definitions [5] . This lack of uniform definitions has been overcome most recently by one of the major advances in antifungal management, i.e. the consensus definitions of the European Organization for Research and Treatment of Cancer (EORTC) and the Mycosis Study Group (MSG) published 2002 [8] . Within the patients with leukemia or lymphoma certain subgroups can now be defined more clearly on the basis of well known and newly described risk factors, i.e. neutropenia >10 days, allogeneic and autologous bone marrow and stem cell transplantation, prolonged corticosteroid therapy, other sustained immunosuppression, graft versus host disease, and concomitant viral infections [9] . Besides individual factors the risk of mould infections depends, e.g. on the concentration of colony forming units in the surrounding air, a factor underlying several influences like the region, season and most of all exposure to construction work [10] .

Fluconazole is the antifungal with the highest number of well-designed prophylaxis trials. In comparative trials oral daily doses have been given from 50 mg up to 400 mg [11, 12] .

The two most relevant trials were placebo controlled and double blinded and involved mainly allogeneic transplant recipients [13, 14] . Fluconazole 400 mg/d was superior to placebo significantly in both the reduction of breakthrough invasive fungal infection and the decrease of attributable mortality. In a longitudinal observation the survival benefit extended beyond the period of fluconazole treatment (75 days) and was accompanied by a lower incidence of intestinal graft versus host disease [15] .

A particular strength of both trials was the homogenous, strictly defined and high risk patient population. Other trialists examined more heterogeneous populations, and thus frequently failed to show a survival benefit of their intervention [12] .

Another large placebo controlled trial on fluconazole 400 mg/d resulted in a significant reduction of proven invasive fungal infection and mortality attributed to fungal infection, but the study population was to heterogeneous to lead to a clear cut recommendation [16] .

There is good evidence (Level A I) that primary prophylaxis with fluconazole 400 mg/d reduces the incidence of invasive fungal infections and the mortality rate in allogeneic bone marrow or stem cell transplant recipients. Itraconazole has a broader spectrum of activity than fluconazole comprising non-albicans Candida species and moulds. Itraconazole capsules result into adequate plasma levels late, if at all, and thus are not recommended. Superior bioavailability is accomplished with itraconazole oral suspension.

A double-blind, double dummy, placebo controlled trial comparing the suspension at a dose of 2.5 mg/kg bid plus nystatin 500.000 IU qid to nystatin alone found a more effective reduction of the rate of fatal candidemia from 2% to zero. Rates of mould infection and death attributed to fungal infection were not decreased [17] .

A trial on randomized allogeneic stem cell transplant recipients compared prophylaxis with intravenous followed by oral itraconazole 400 mg/d versus fluconazole 400 mg/d given for until day 100 post transplant. Itraconazole reduced proven invasive fungal infections more effectively, but failed to significantly improve the fungal death rate [18] . The study has been criticized for being underpowered due to the inclusion of only 140 patients. Another controlled trial compared intravenous itraconazole 200 mg/d or oral suspension 7.5 mg/kg/d with parenteral or oral fluconazole 400 mg/d i.v. The trial included 297 allogeneic transplant recipients. In itraconazole recipients a statistically significant reduction of breakthrough invasive fungal infections was accomplished, but went along with a higher rate of liver and kidney toxicity leading to a 36% withdrawal rate [19] . Meta-analyses referring to the usefulness of itraconazole prophylaxis render conflicting results so far [20, 21] . The use of itraconazole demands regular plasma level monitoring to evaluate whether plasma concentrations of >500 ng/ml are reached and maintained. Itraconazole can effectively reduce breakthrough fungal infections, but does not reduce attributable mortality rates in clinical trials.

Posaconazole has a broad spectrum of activity comprising Candida spp, Aspergillus spp, zygomycetes and Fusarium spp. The oral solution is readily absorbed in patients with hematological diseases [22] . Posaconazole prophylaxis at a dose of 3200 mg/d has been evaluated in two large welldesigned randomized controlled clinical trials.

The first international multicenter and randomized double-blind trial focused on the prophylaxis in a high risk population of allogeneic transplant recipients. Six-hundred patients were enrolled into this study. The control group received fluconazole. The group receiving posaconazole had a significant lower incidence of invasive fungal disease than the control group. Not only was posaconazole adequately absorbed in this patient population, it also demonstrated a significant survival benefit in regards to mortality caused by invasive fungal disease [23] . The results of this study advocated the prophylactic use posaconazole in patients with severe GVHD after allogeneic stem cell transplantation.

The second trial enrolled 602 patients with a first chemotherapy cycle for AML remission induction. Remarkably, this population is the most homogenous examined in an antifungal prophylaxis trial, so far. The trial has been difficult to design. The above documents that there is no evidence based standard approach for this population, thus defining an unmet medical need. However, the majority of the clinicians involved in this trial were already using prophylactic fluconazole or itraconazole in AML induction I patients. So the comparator arm was the standard approach chosen by the single trial center out of these two azoles.

Posaconazole reduced the rates of breakthrough invasive fungal infections and besides other survival endpoints, the overall mortality until d100 was significantly decreased [24] . This leads to a recommendation to use posaconazole prophylaxis in these patients (Level AI). These results could mark the end of a decade long debate on the general feasibility of a significant reduction of overall mortality by a prophylactic antifungal in hematological patient populations. Micafungin 1 mg/kg was compared to fluconazole 400 mg in a trial recruiting 882 patients undergoing autologous and allogeneic stem cell recipients with various underlying malignant diseases. Invasive candidiasis was effectively prevented by both regimens, but micafungin was more effective against invasive aspergillosis. No significant reduction of the overall and attributable fungal mortality was detected [25] . The results are difficult to put into the context of other trials, since for the 46% autologous transplant patients both treatments are equally experimental [26, 27] .

An improved rate of mortality due to fungal infection has been shown for fluconazole 400mg/d in allogeneic transplant recipients (Level AI). Furthermore in patients with higher grade GvHD posaconazole 3200 mg/d effectively prevented breakthrough invasive fungal infections and reduced attributable mortality (Level AI). Overall mortality as well as breakthrough fungal infections have been reduced by posaconazole prophylaxis in long-term neutropenic patients undergoing AML induction chemotherapy (Level AI). Micafungin may be used in allogeneic stem cell transplant patients to prevent aspergillosis. Invasive fungal infections are an ongoing diagnostic and prognostic dilemma. The principal efficacy of antifungal prophylaxis has been proven for the patient groups at highest risk. Further intensive efforts need to be undertaken to decrease the incidence and mortality of invasive fungal infections.

Invasive fungal infections have evolved into important causes of morbidity and mortality in patients with severe underlying diseases. For more than three decades, treatment has been limited to amphotericin B deoxycholate with or without flucytosine. Therapeutic options only emerged with the clinical development of fluconazole and itraconazole in the late 1980s. The past ten years, however, have witnessed a major expansion in our antifungal armamentarium through the introduction of less toxic formulations of amphotericin B, and, more recently, the development of improved antifungal triazoles and echinocandin lipopeptides [6] . This article summarizes the clinical pharmacology of caspofungin, voriconazole and posaconazole.

The echinocandin lipopeptide caspofungin is the first of a new class of antifungal compounds that inhibit the synthesis of 1,3-beta-D-glucan. This homopolysaccharide is a major component of the cell wall of many pathogenic fungi and absent in mammalian cells. It provides osmotic stability and is important for cell growth and cell division. In vitro, caspofungin has broadspectrum antifungal activity against Candida-and Aspergillus spp. without cross-resistance to existing agents. The compound exerts prolonged postantifungal effects and fungicidal activity against Candida species and causes severe damage of Aspergillus fumigatus at the sites of hyphal growth. Animal models have demonstrated efficacy against disseminated candidiasis and dissemi-nated and pulmonary aspergillosis, both in normal and in immunocompromised animals [7] .

Caspofungin is only available for intravenous administration. The compound exhibits dose-proportional plasma pharmacokinetics with a beta half-life of approximately 15 hours that allows for once daily dosing. It is highly (>95%) protein bound and distributes into all major organ sites including the brain; however, concentrations in uninfected CSF are low. Caspofungin is metabolized by the liver following degradation and is slowly excreted into urine and feces; only small fractions (<2%) of a dose are excreted into urine in unchanged form [7, 25] .

At the current dosage, caspofungin is generally well tolerated, and only a small fraction of patients enrolled on the various clinical trials ( 7 days, the response rate was 56% [14] . Finally, in a large, randomized, double blind clinical trial including 1095 patients, caspofungin was as effective as liposomal amphotericin B for empirical antifungal therapy in persistently febrile granulocytopenic patients but better tolerated. The proportion of patients who survived at least seven days after therapy was greater in the caspofngin group (92.6 vs. 89.2%) [32] .

Currently, caspofungin is licensed in the European Union and the United States in patients 18 years of age for second line therapy of definite or probable invasive aspergillosis, for primary therapy in non-neutropenic patients with invasive Candida-infections, and for empirical antifungal therapy in granulocytopenic patients with persistent fever. The recommended dose regimen consists of a single 70-mg loading dose on day 1, followed by 50 mg daily thereafter, administered over one hour. No dosage adjustment is required in patients with renal insufficiency. In patients with mild hepatic insufficiency (Child-Pugh category A), no adjustments are needed; in patients with moderate hepatic insufficiency (Child-Pugh category B), decreasing the maintenance dose to 35 mg/day is recommeded after the loading dose of 70mg. No recommendations exist for patients with severe hepatic insufficiency (Child-Pugh category C) [7] .

Voriconazole is a recently approved synthetic antifungal triazole with activity against a wide spectrum of clinically important yeasts and moulds, including Candida spp., Cryptococcus neoformans, Aspergillus and other hyaline moulds, dematiaceous moulds as well as dimorphic moulds, both in vitro as well as in animal models. A notable exemption are the zygomycetes, against which voriconazole is intrinsically inactive. Similar to itraconazole, voriconazole is generally considered fungistatic against Candida but fungicidal against Aspergillus spp [5, 11] .

Voriconazole is available in oral and intravenous formulations. Following oral administration, peak plasma levels occur within 1 to 2 hours, and bioavailability exceeds 90% in the fasted state. The compound has nonlinear pharmacokinetics possibly resulting from saturable first-pass metabolism. Plasma protein binding is 58%, and the mean volume of distribution accounts for 2L/kg. Tissue and CSF levels exceed those of trough plasma levels severalfold. The plasma half-life is 6 hours, with elimination primarily occurring by oxidative hepatic metabolism to at least 8 metabolites that are eliminated via the urine; less than 2% of a dose of VCZ are excreted unchanged in urine. The major isoenzyme involved in VCZ metabolization is CYP2C19, but CYP2C9 and CYP3A4 also contribute. There is a wide between-subject variability in the disposition of VCZ, that is related to genetic CYP2C19 polymorphism [5, 20] .

Voriconazole has an acceptable safety profile. The accrued clinical data indicate that side effects include four distinct clinical categories: Transient liver enzyme abnormalities (10-20%), skin reactions (<10%), hallucinations or confusion (<10%) and transient, dose-related visual disturbances (altered or enhanced perception of light, blurred vision; 25-45%) [5] . However, drug-related adverse effects requiring the discontinuation of voriconazole were infrequent in comparative clinical trials (2-13%) [1, 10, 31] . Voriconazole is both substrate and inhibitor of CYP2C19, CYP2C9, and CYP3A4. VCZ significantly increases exposure to ciclosporin, tacrolimus, benzodiazepins, vinca-alkaloids, the statins, omeprazole,warfarin, sulfonylurea drugs, phenytoin,protease inhibitors other than indinavir, non-nucleoside reverse transcriptase inhibitors, requiring dosage adjustment and/or monitoring. VCZ exposure is significantly decreased by phenytoin, rifabutin, carbamazepine, rifampin, phenobarbital. Concurrent use of the three latter enzyme-inducers with VCZ is contraindicated (risk subtherapeutic levels of VCZ) as is the concurrent use of terfenadine, astemizole, cisapride, quinidine, pimozid (risk of QTc-prolongation due to increased exposure of these agents), ergotamin (risk of ergotism due to increased exposure) and sirolimus (increased exposure). Dosage adjustment of VCZ is necessary when it has to be used concurrently with phenytoin or rifabutin [5] .

Voriconazole has demonstrated excellent clinical efficacy in phase II and III clinical trials in patients with oropharyngeal candidiasis [9] and esophageal candidiasis [1] . In salvage studies of invasive aspergillosis and other mycoses, responses were observed in 41 to 55% of patients [3, 19] . A multinational, randomized phase III clinical trial of voriconazole and conventional amphotericin B followed by other licensed antifungal therapy for primary therapy of invasive aspergillosis revealed superior antifungal efficacy and improved survival of voriconazole treated patients at week 12 [10] . A randomized comparative study of voriconazole versus conventional amphotericin B followed by fluconazole for treatment of candidemia in non-neutropenic patients showed similar response rates and end of treatment and similar survival at three months [12] . In a large international collaborative study of voriconazole versus liposomal amphotericin B for empirical therapy, voriconazole did not meet the prespecified statistical endpoint for non-inferiority in a composite endpoint but was associated with significantly fewer breakthrough invasive fungal infections, particularly those due to invasive aspergillosis [31] . Finally, several reports also suggest the potential usefulness of VCZ for treatment of infections by unusual hyaline and dematiaceous fungi [19] , and for treatment of cerebral mould infections [23] .

Voriconazole has been approved in the United States and the European Union for treatment of invasive aspergillosis, fusariosis, and scedosporiosis, and for primary treatment of invasive candidiasis in non-neutropenic patients. The recommended IV dosages for adult and pediatric patients >2 years of age are 6mg/kg BID on day 1, followed by 4 mg/kg BID. The oral dosages in adults are 400mg BID on day 1 (<40kg: 200mg BID), followed by 200mg BID (<40kg: 100mg BID); recommended oral dosages in pediatric patients are similar to IV dosages. However, children may need higher dosages to reach the same exposure as adults [33] . In patients with renal insufficiency, no dosage adjustment is needed for the PO formulation; because of the renal clearance of the intravenous carrier, patients with a creatinine clearance of <50mL/min should receive voriconazole by the oral route. In patients with mild to moderate hepatic function abnormalities (Child-Pugh category A and B), half of the daily maintenance dosage is recommended after the initial loading dose. Recommendations for severe liver failure (Child-Pugh category C) are lacking [11] .

Posaconazole is a novel lipophilic antifungal triazole that, similar to other members of this class, inhibits CYP450-dependent 14-alpha demethylase in the biosynthetic pathway of ergosterol. This interaction leads to an accumulation of toxic 14-alpha methylsterols and a depletion of ergosterol, resulting in a perturbation of the function of the fungal cell membrane and blockage of cell growth and replication. In vitro, posaconazole has potent and broad-spectrum activity against opportunistic, endemic, and dermatophytic fungi. This activity extends to organisms that are often refractory to existing triazoles, amphotericin B or echinocandins such as C. glabrata, C.krusei , A.terreus, Fusarium spp. In vivo, a large variety of animal models of invasive fungal infections has provided consistent evidence of therapeutic efficacy against these organisms, both in normal and in immunocompromised animals. Importantly, posaconazole also possesses activity against zygomycetes both in vitro and in vivo, distinguishing it from all available azoles [8] .

The compound is available as oral suspension only and achieves optimal exposure when administered in 2 to 4 divided doses given with food or a nutritional supplement. The compounds has a large volume of distribution in the order of 5 L/ kg and a prolonged elimination half-life of approximately 20 hours. Posaconazole is not metabolized through the cytochrome P450 enzyme system but primarily excreted in unchanged form in the feces. It is inhibitory against cytochrome P3A4, but has no effects on 1A2, 2C8, 2C9, 2D6 and 2E1 isoenzymes, and therefore, a limited spectrum of drug-drug interactions can be expected [4, 13] .

Posaconazole appears to be well tolerated in a manner comparable to fluconazole. The overall safety of posaconazole has been assessed in more than 400 patients with invasive fungal infections from two open label clinical trials [18] . Treatment-related adverse events occurred in 38% of patients (164/428); the most common were nausea (8%), vomiting (6%), headache (5%), abdominal pain (4%), and diarrhea (4%). Treatment-related abnormal liver function test results were observed in up to 3% of patients. There were no clinically significant differences in mean QTc interval change from baseline. Serious adverse events considered possibly or probably related to posaconazole occurred in 35 (8%) patients. The most common severe adverse events were altered drug level, increased hepatic enzymes, nausea, rash, and vomiting (1% each). The drug-drug interaction potential of posaconazole has been investigated in seven open label, cross-over drug interaction studies. No dose adjustments are needed when posaconazole is coadministered with glipizide, zidovudine, and lamivudine, whereas dosages of ritonavir and indinavir may need to be lowered. Monitoring of cyclosporine and tacrolimus blood concentrations is mandatory, and dose adjustments should be made accordingly. Due to a relevant decrease in posaconazole concentrations and the associated risk of subtherapeutic plasma concentrations, concomitant use with rifabutin, phenytoin, or cimetidine should be avoided. As with other azoles, caution is advised when posaconazole is coadministered with CYP3A4 substrates that have the potential to prolong the QTc interval [8] .

Apart from two phase II clinical trials for first- [28] and second line [24] therapy of HIV-associated oropharyngeal and esophageal candidiasis, preliminary results have been presented for the pivotal phase II salvage study in patients with possible, probable and proven invasive fungal infections refractory to or intolerant of standard therapies [21] and a phase III randomized clinical trial comparing posaconazole to fluconazole for treatment of oropharyngeal candidiasis [17] . Posaconazole has demonstrated strong antifungal efficacy in phase II and III clinical trials in immunocompromised patients with oropharyngeal and esophageal candidiasis. Posaconazole also showed promising efficacy as salvage therapy in a large phase II study including 330 patients with invasive fungal infections intolerant to or refractory to standard therapies and a contemporaneous external control of 279 patients [21] . Most patients (86%) were refractory to previous therapy. Successful outcomes at end of treatment in the posaconazole and in the contemporaneous external control cohorts were 42 vs. 26% in aspergillosis (107 and 86 pts.), 39 vs. 50% in fusariosis (18 vs. 4 pts.), 56 vs. 50% in zygomycoses (11 vs. 8 pts.) , 69 vs. 43% (16 vs. 7 pts.) in coccidioidomycosis, 52 vs. 53% in candidiasis (23 vs. 30 pts), 48 vs. 58% in cryptococcosis (31 vs. 64 pts.) , 81 vs. 0% in chromoblastomycosis (11 vs. 2 pts.) , and 64 vs. 60% in other invasive fungal infections (30 vs. 20 pts.) . Preventative phase III studies in high risk patients with hematopoietic stem cell transplantation and acute leukemias have been completed [27] , and a phase II study in patients with non-meningeal coccidioidomycosis is ongoing.

Posaconazole has recently been approved in the European Union for treatment of aspergillosis, fusariosis, chromoblastomycosis and coccidioidomycosis refractory to or intolerant of standard theraüies. The recommended daily dosage for salvage treatment is 400 mg BID given with food; for patients not tolerating solid food, a dosage of 200 mg QID is recommended, preferentially given with a nutritional supplement. Current data indicate no need for dosage adjustments based on differences in age, gender, race, renal or hepatic function [8] .

The availability of alternative therapeutic options is an important advance in the supportive care of immuncompromised patients at high risk for invasive fungal infections. At the same time, however, antifungal therapy has become increasingly complex. In addition to information on prior antifungal therapies, microbiological data, existing co-morbidities and co-medications, a detailed knowledge of the available antifungal armamentarium and contemporary clinical trials is needed more than ever when selecting the optimal management strategy for the individual patient. 

The introduction of Rituximab R) into the treatment of malignant lymphomas of the B cell lineage has had a major impact on the management of these diseases. In the two most frequent lymphoma subtypes, the diffuse large B cell lymphomas (DLBCL) and the follicular lymphomas (FL), several multicenter prospective randomized trials consistently demonstrated an improved outcome when Rituximab was added to chemotherapy (1, 3) . Significant increases in response rates, response duration and even in overall survival by Rchemotherapy were also observed in FL in first and second line therapy (2, 5, 6, (8) (9) (10) .

In both lymphoma entities it was also shown that further Rituximab maintenance in remission was able to prolong response duration when initial chemotherapy did not include Rituximab (2, 7) .

So far, the question remains unanswered, however, whether Rmaintenance is also effective when remission is induced by a Rituximab containing chemotherapeutic protocol. In order to address this question the German Low Grade Lymphoma Study Group (GLSG) embarked on a prospective randomized comparison of R-maintenance versus no further treatment in patients with relapsed or refractory FL or mantle cell lymphoma (MCL) responding to salvage therapy with Rituximab in combination with Fludarabine, Cyclophosphamide and Mitoxantrone (R-FCM (2) .

This study included patients of ages 18 years and older with relapsed or refractory follicular or mantle cell lymphoma according to the WHO classification (4) .

Patients underwent an initial randomization for FCM versus R-FCM (Rituximab 375 mg/m/d day 0, Fludarabine 25 mg/m/d days 1-3, Cyclophosphamide 200 mg/m/d days 1-3 and Mitoxantrone 8 mg/m/d day 1). Patients achieving a complete or partial remission underwent a second randomization for two courses of Rituximab (375 mg/m/d at 4 consecutive weeks) to be given 3 and 6 months after completion of salvage therapy.

One hundred and ninety-five patients were enrolled into the trial. In June 2001 the applied one sided sequential test showed a significant advantage for the R-FCM arm over FCM alone and further randomization for this question was stopped after 147 patients (2) . All subsequent cases received R-FCM. Randomization for R-maintenance versus no further treatment continued until May 2005 when the respective statistical test showed a significant advantage for the R-maintenance arm. 174 cases are presently evaluable for response to therapy, response duration and toxicity and 138 had received R-FCM initially. A significantly longer response duration was observed for R-maintenance for the total group of patients and also for patients having received R-FCM initially with the median not being reached as compared to a median of 17 months only for patients receiving no further treatment (p=0.0024). When restricting the analysis to patients with FL only who were uniformly treated with R-FCM for salvage therapy (n=81) a significant prolongation of response duration by R-maintenance therapy as compared to observation only was observed with the median not being reached as to a median of 23 months (p=0.035) ( Figure 1 ).

When applying this analysis to patients with MCL and uniform R-FCM therapy (n=47) a beneficial effect of R-maintenance was also observed. Although the median response duration did not differ substantially between Rmaintenance and observation only, the proportion of ongoing remission at 4 years was 30 % on R-maintenance as compared to 0 % on the observation only arm (p=0.048) (Figure 2 ).

The current study clearly indicates that maintenance therapy with Rituximab leads to a significant improvement of response duration in patients with relapsed or refractory follicular lymphoma and, to a lesser degree, also in patients with mantle cell lymphoma who responded to salvage therapy comprising a Rituximab-chemotherapy combination. These data show a sustained efficacy of Rituximab through all phases of treatment and demonstrate that its antilymphoma activity in remission is not compromised by adding Rituximab to prior chemotherapy.

These results were most recently confirmed by a study carried out by an EORTC intergroup study which used a similar trial design (10) . Hence, patients with relapsed FL were initially randomized for salvage therapy with CHOP versus R-CHOP and subsequently underwent a second randomization for no further treatment versus Rituximab maintenance consisting of Rituximab 375 mg/m2/d to be given as a single infusion every 3 months for a total of 2 years. A subgroup analysis of 189 cases who had uniformly received R-CHOP for remission induction revealed a significantly longer median response duration for patients on the R-maintenance arm of 51 months as compared to 15 months for patients receiving no further treatment (p<0.0001)(van Oers, ASH 2005). Hence, both studies consistenly reveal a long lasting beneficial effect of Rituximab maintenance on response duration after salvage therapy with different Rituximab containing regimens.

Whether R-maintenance is also effective in first line therapy after Rchemotherapy combinations and whether the application of Rituximab during all phases of therapy might be superior to restricting the addition of Rituximab either to initial chemotherapy or to maintaining remission after first line chemotherapy without Rituximab remains currently unanswered. 

Farnesyltransferase inhibitors (FTIs) represent a new class of signal transduction inhibitors that block the processing of cellular polypeptides with a cysteine terminal residues and, by so doing, interdict multiple pathways involved in proliferation and survival of diverse malignant cell types. Tipifarnib is an orally bioavailable, nonpeptidomimetic FTI that is being tested clinically in diverse hematologic malignancies including acute myelogenous leukemia (AML). Tipifarnib induces overall response rate of 25% with durable complete remissions (CRs) in 15% of elderly adults (> 65 years), including those with secondary AML and/or adverse cytogenetics. FTI therapy is accompanied by a relatively low toxicity profile, thereby providing an important alternative to traditional cytotoxic approaches for elderly patients who are not likely to tolerate or even benefit from aggressive chemotherapy. The roles for FTIs in the treatment armamentarium for myeloid malignancies continue to evolve. Critical questions have yet to be answered, for example, questions pertaining to the optimal dose level and schedule, the disease subgroups most likely to respond therapeutically, and appropriate combinations with standard cytotoxics and new biologics. Gene profiling studies of malignant target cells obtained during FTI clinical trials are critical to our ability to identify patients who are likely to respond to FTIs and to develop mechanisms for overcoming FTI resistance in a clinically meaningful fashion. The clinical trials and correlative laboratory studies that are currently in progress and under development will provide the critical foundations for defining the optimal roles of FTIs in patients with myeloid malignancies.

Acute myelogenous leukemia (AML) affects older adults disproportionately, with a median age at diagnosis of 68. Long-term survival rates are low in elderly AML patients, with median survival less than 1 year [9, 17] . In addition, the rates of chemotherapy-associated severe toxicities, along with death, are very high in this disease. In adults over age 60, the risk of treatment-related death approaches 20% or more during the induction phase of therapy [1, 17] . Even for those patients who achieve complete remissions (CRs) through chemotherapy, the remission duration is generally on the order of only 6-9 months [1, 9, 17] . Adverse karyotypic profiles, including deletions of chromosomes 5 and 7, abound in elderly AML patients, and portend poor long-term outcomes [5] . Chemotherapy failure in elderly patients with AML is also attributable to the presence of multi-drug resistance phenotype, which correlates negatively with remission rate [11] . For all of these reasons, new agents with novel mechanisms of action and fewer non-hematologic toxicities are necessary to improve outcomes in patients with AML.

Farnesyltransferase inhibitors (FTIs) represent a new class of signal transduction inhibitors that impede critical cell growth and survival signals. These agents are potent and selective competitive inhibitors of farnesyltransferase (FT), an intracellular enzyme that catalyzes the transfer of a farnesyl moiety to the cysteine terminal residue of a substrate protein. A host of intracellular proteins are substrates for prenylation via FT, including Ras, Rho-B, Rac, membrane lamins, and centromeric proteins that interact with microtubules to promote the completion of mitosis [3, 15, 16] . Interruption of prenylation may prevent substrates from undergoing maturation which, in turn, may result in the inhibition of cellular events that depend on the function of those substrates. The finding that FT is a biologically relevant target and the antitumor activity in preclinical models that results from FT inhibition are the impetus for the development of FTIs in the clinical arena. Hematologic malignancies provide a fertile testing ground for such agents because of the relative ease with which tumor tissue can be obtained throughout the therapeutic course. The ability to obtain target tissue in a longitudinal fashion provides a unique opportunity to define the relevant molecular components that may be modulated by these compounds and to relate those molecular effects to the clinical outcome.

Clinical Trials in Adults with Acute Myelogenous Leukemia (AML) The first clinical testing of FTIs in AML was a Phase I trial of the orally bioavailable FTI Tipifarnib administered for 21 days in patients with relapsed or refractory AML [7] . Consistent inhibition of FTase activity occurred at or above 300 mg BID orally and dose-limiting toxicity (DLT), manifested as readily reversible central neurotoxicity, was observed at 1200 mg BID. Oral absorption was rapid, with linear pharmacokinetics, and there was a dose-dependent increase in drug concentration in marrow with sustained levels 2-3-fold higher than concomitant levels in peripheral blood. Clinical responses were observed in 10 of 34 patients (29%), including 2 CRs in patients with relapsed AML, and occurred across all dosing levels (100 mg BID -900 mg BID) without strict relationship to the degree of leukemic cell FTase inhibition. Intriguingly, responses were independent of ras mutational status, as none of the 34 leukemic samples demonstrated an N-ras mutation.

Based on these findings, Lancet, et al [10] conducted a Phase II study of Tipifarnib 600 mg BID for 21 out of 28-63 days in elderly, previously untreated patients with AML, enrolling 171 patients (158 AML, 13 high-risk MDS/CMML; median age 73) who refused or were poor candidates for standard cytotoxic induction chemotherapy. Of the AML patients, 136 were > age 65 with MDS/AML in 82% and adverse cytogenetics in 49%. A CR rate of 15% was attained in these poor-risk AML patients, including patients with adverse cytogenetics (e.g., -5, -7, 11q23 abnormalilties,-20q, complex). Partial remissions (PR) or hematologic improvement (HI) occurred in an additional 10%, for an overall response rate of 25%. Median CR duration was 32 weeks, and achievement of CR appeared to impart an overall survival advantage compared with non-CR (62 wks vs 19 wks). Importantly, treatment-related mortality was less than 10%, and the majority of patients did not require hospitalization. The most frequently observed nonhematologic toxicities included infection/febrile neutropenia, gastrointestinal events (diarrhea, amylase/lipase elevation), neurologic toxicity, and skin rash. Adverse cytogenetics and age > 75 appeared to predict for poorer survival in a multivariate analysis. Measurements of inhibition of farnesylation of the chaperone protein HDJ-2 in marrow blasts obtained on day 8 of Tipifarnib therapy revealed an increase in unfarnesylated protein in 75% of marrow samples, while inhibition of farnesylation of the nuclear membrane protein Lamin A could be detected in 92% of all concomitantly-obtained samplers of buccal mucosa [10] . This provocative finding may provide insight into potential mechanisms of FTI resistance.

The roles for FTIs in the treatment armamentarium for myeloid malignancies continue to evolve. Critical questions have yet to be answered, for example, questions pertaining to the optimal dose level and schedule, the disease subgroups most likely to respond therapeutically, and appropriate combinations with standard cytotoxics and new biologics. The Southwest Oncology Group is conducting a multi-armed study of Tipifarnib for previously untreated patients with AML who are > 70 years to examine different dosing levels (300 mg vs 600 mg BID) and intervals (21 out of 28 days vs. every other week) with respect to efficacy and toxicity parameters. To date, CRs and/or PRs have been seen in all four arms without unexpected or dose-limiting toxicities.

To date, post-remission chemotherapy for AML in elderly patients or those with other poor-risk features (e.g., MDS/AML, treatment-related AML, adverse cytogenetics) has not prolonged disease-free or overall survival (DFS, OS) [1, 5, 9, 17] . In this group of patients, median CR duration is < 8 months and 1 year DFS is < 20%. Given that Tipifarnib has clinical activity in poor-risk AML in elderly adults, and is both easily administered and well-tolerated, it is logical to evaluate the feasibility and potential efficacy of maintenance Tipifarnib monotherapy in prolonging CR and DFS in adults with AML with poor-risk features in first CR following induction and consolidation therapies [8] . To date, Tipifarnib 400 mg BID has been given for 14 out of 21 days for up to 16 cycles (48 weeks) to 38 adults with AML with multiple poor-risk features (median age 64, secondary AML in 34%, adverse cytogenetics in 47%). Transient and modest myelosuppression was common but rarely resulted in any adverse events, including transfusions and hospitalizations, and resolved either spontaneously or with dose reduction. In 34 evaluable patients, median DFS is 14 months (64% DFS > 8 months, 57% DFS >12 months, 33% DFS >18 months). For patients with secondary AML, 7/10 have DFS > 12 months and 6 continue in CR 7+-28+ months (median 22+). For patients with adverse cytogenetics, 9/15 have DFS > 12 months and and 6 continue in CR 6+-39+ months (median 23+ months). Importantly, for patients who relapse, Tipifarnib maintenance does not appear to have a negative impact on the ability to achieve a second CR [8] . These data are a platform for a definitive Phase III trial.

The full development of FTIs in the therapeutic armamentarium for hematologic (and other) malignancies will require the design and testing of rational combinations of FTIs with cytotoxic, biologic and immunomodulatory agents in both the laboratory and the clinic. In this regard, Cortes, et al [2] are examining the addition of Tipifarnib 300 mg BID for 21 days to ara-C plus idarubicin induction and consolidation, followed by Tipifarnib maintenance 300 mg BID for 14 days every 4-6 weeks for 6 months. Early results demonstrate a 72% CR rate in the first 23 patients, including older adults and patients with adverse cytogenetics, with reversible diarrhea and hyperbilirubinemia being transient side effects. In another design, Tipifarnib is being combined with oral VP-16 for adults > age 70 years with newly-diagnosed AML, with dose escalation of both the Tipifarnib (300, 400, 600 mg BID) and VP-16 (100, 150, 200 mg daily days 1-3 and 8-10) and examination of two schedules of Tipifarnib (14 days vs. 21 days every 28-63 days) [Karp JE, unpublished observastions] .

Gene Expression: Insights into FTI Mechanisms of Action and Determinants of Response Maximizing the efficacy of new therapies, including FTIs, in the clinical arena will ultimately depend upon an improved understanding of the disease biology to which the agent may be directed. Toward this end, gene expression profiling through microarray technology may serve to identify genes and gene expression signatures that could predict for clinical response to in vivo treatment with FTIs. Raponi, et al [12] identified integrated gene networks whose activities are modulated in an orchestrated fashion to yield net cell death in diverse AML cell lines and in primary AML marrow samples. Further, these investigators conducted gene profiling of marrow blasts from adults with relapsed and refractory AML obtained prior to treatment with Tipifarnib in the setting of an international Phase II study in relapsed and refractory AML patients [6, 13] . In this setting, it appears that 8 genes are differentially expressed in responders vs. nonresponders, with overexpression of one gene in particular, the lymphoid blast crisis oncogene (oncoLBC or AKAP13), capable of accurately predicting clinical response to Tipifarnib [13] . Provocatively, the AKAP13 protein acts a guanine nucleotide exchange factor for Rho proteins [18, 19] contains a region that is homologous to an -helical domain known to interact with nuclear envelope protein lamin B [4] . This is especially intriguing because Rho and lamin proteins are farnesylated, and AKAP13 activates both types of proteins. Most recently, Raponi and colleagues have analyzed gene expression profiles of 79 marrow blast samples from elderly adults with newly diagnosed AML obtained prior to, during and/or following Tipifarnib treatment on the Phase II study discussed above. Tipifarnib treatment resulted in changes in global gene expression that persisted for up to 120 days following treatment, with roughly 500 genes involved in protein biosynthesis, intracellular signaling, DNA replication and cell cycle progression undergoing significant changes in expression as a result of in vivo exposure to Tipifarnib. A subset of 27 genes appears to be differentially expressed in responders vs. nonresponders. Moreover, when the gene expression signatures identified in relapsed/refractory patients were tested in pretreatment marrow blast samples from newly diagnosed patients, a 6-gene combination was found to have predictive potential for eventual response.

In summary, while we are beginning to elucidate mechanisms by which transformed cells respond to FTIs and the optimal settings in which they do so, the precise mechanisms remain to be defined. The original notion that these agents targeted Ras mutations is clearly not complete, and it is likely that FTI's have an impact on multiple molecules and pathways involved in cellular integrity [12] [13] [14] 16] . Studies to define the mechanisms by which FTIs alter cellular metabolism and modulate the activities of specific signaling pathways in both normal and malignant marrow precursors are a pivotal part of this effort. Gene profiling studies of malignant hematopoietic cells obtained during FTI clinical trials are critical to our ability to identify patients who are likely to respond to FTIs and to develop mechanisms for overcoming FTI resistance in a clinically meaningful fashion. The clinical trials and correlative laboratory studies that are currently in progress and under development will provide the critical foundations for defining the optimal roles of FTIs in patients with myeloid malignancies. 

A prevalence of 30% of invasive fungal infections (IFI) is observed in patients treated with cytotoxic chemotherapy for hematological malignancies followed by severe and long lasting neutropenia. Despite substantial advances in the last decade, invasive fungal infections are still associated with a mortality of up to more >70%, in particular for those patients suffering from invasive aspergillosis. There has been made some diagnostic progress with the advent of new nonculture based tools. However, response rates of first-line invasive are about 40 to 60% in the first-line therapy of invasive fungal infections. Salvage therapy will still be necessary for those being intolerant or refractory with their IFI. Following a long period of stagnation, there has been considerable progress during the last five years in the treatment of refractory invasive fungal infections. This review highlights new treatment options for the salvage setting of invasive fungal infections in cancer patients. Voriconazole, a second generation azole, is considered to be the new gold standard for first line treatment of invasive aspergillosis. The starting dose is 6 mg/kg twice daily on day 1 followed by 4 mg/kg twice daily on subsequent days. Voriconazole administered in an intravenous/oral sequence is superior to Amphotericin B (1.0 mg/kg) in terms of response rates (52.8% vs. 31.6%) and overall 12-week survival (70.8% vs. 57.9%). This superiority to amphotericin B was demonstrated both for isolated pulmonary involvement and for extrapulmonary involvement, in patients with and without neutropenia, and in allogeneic stem cell transplant recipients [1] . Responses ranging from 16% to 54% have even been shown in subjects with cerebral involvement, which is otherwise associated with a very poor prognosis [2] . Transient disturbed vision is the most common side effect of voriconazole (observed in approximately 30%).

Caspofungin a new echinocandin was approved 2001 for 2nd-line Therapy of invasive aspergillosis in refractory or intolerant patients [3] . Caspofungin showed a favorable safety profile, with adverse event noted in a minority of patients. Micafungin has already been approved salvage treatment of invasive aspergillosis in some countries including Japan. In an open-label phase II trial investigated micafungin (FK-463) in 283 patients with proven or probable invasive aspergillosis (alone or in combination with other antifungals) at dose levels of 75 mg/day or 1.5 mg/kg (in subjects weighing less than 40 kg). The trial included 81 patients (29%) with neutropenia. Observed response rates stratified by risk factors (underlying disease, allogeneic transplantation, FK-463 as singleagent or combination treatment), ranged from 22% for allogeneic transplant recipients (n = 49) and 49% for leukemia patients (n = 45) [4] .

Posaconazole is another broad spectrum azole that was recently licensed for 2nd-line therapy of invasive aspergillosis [5, 6] . Walsh et al. presented the results of a multicenter study investigating posaconazole as second-line therapy (refractory or intolerant to amphotericin B or itraconazole) at the ASH 2003 meeting. The posaconazole arm contained individual patients who had already received voriconazole or caspofungin. An expert panel retrospectively compared the 107 posaconazole therapies with 86 carefully collected control cases who had been treated during the same period with similar pathogens in similar hospitals, but none of the control patients had been previously treated with voriconazole or caspofungin. The groups were otherwise comparable in terms of demographics. Neutropenia (ANC < 500/l) was present at study enrollment in 20% of the posaconazole patients and 30% of control patients. Overall response at the end of treatment was 42% in the posaconazole arm (including 36% PR) and 26% in the control group (including 16% PR) (odds ratio = 4.06 (95% CI = 1.50, 11.04) p=0.006). Response was 39% in subjects with pulmonary aspergillosis (25% in the control group), and 53% in subjects with primary extrapulmonary involvement (45% in the control group). Five out of 21 neutropenic subjects (24%) in the posaconazole group responded to treatment versus 2 out of 26 neutropenic subjects in the control arm (8%). Response rates for posaconazole were also better in the subgroup of allogeneic transplant recipients (15/48 cases (31%) for posaconazole vs. 7/34 cases in the control group (21%) [7] .

Thirty-nine out of 107 patients in the posaconazole group were rated nonresponders versus 52 out of 86 patients (60%) in the control group. Kaplan-Meier analysis disclosed a survival benefit for the posaconazole group (p<0.001, log-rank test). The limitation of the presented study is a case control design, albeit with a complex design. The efficacy of posaconazole in invasive aspergillosis needs to be confirmed in a 1st-line prospective controlled trial in IFI.

Combination of antifungal drugs has been reported in the salvage setting mainly with a combination of polyenes and echinocandins [8] . This attempt is more and more used in the common practice for refractory patients. Caspofungin has been combined with amphoerticin B, itraconazole or voriconazole in an open label non comparative trial (n=53) for refractory (87%) or intolerant (13%) patients. Maertens reported a success rate (end of therapy) of 55% which dropped to 49% after 12 weeks [9] . Although safety of antifungal combination therapy seems to be not a major issue, efficacy of this approach has not been proved in a prospective controlled trial. Treatment for acute myelogenous leukemia (AML) is one of the most intensive treatment modalities in oncology, in particular in the pediatric population. It has been shown that infection-related morbidity and mortality is unacceptably high among these patients, in particular during chemotherapy-induced neutropenia, which is the most important single risk factor for bacterial and fungal infections.

Early studies have shown that the incidence of microbiologically documented infections in febrile neutropenic cancer patients approached 100% in patients untreated for up to 3 weeks.1 In response to the poor outcome observed in the majority of these patients, early use of empirical antibacterial and antimycotic therapy has evolved and dramatically decreased mortality. 2 The characterization and purification of hematopoietic growth-factors, a class of cytokines that regulate proliferation, differentiation, and function of hematopoietic cells, offered the promise of a new dimension for supportive care in the cancer patient. Among them, granulocyte colony-stimulating factor (G-CSF) regulates the production of the neutrophil lineage. 3 The administration of G-CSF to humans results in a dose-dependent increase in circulating neutrophils, mainly because of a reduced transit time from stem cell to mature neutrophils. In addition, G-CSF enhances phagocyte function, such as superoxide generation and fungicidal activity. Numerous studies have evaluated the prophylactic use of G-CSF in both pediatric and adult patients undergoing therapy for cancer.4,5 Unfortunately, the results of the studies performed in this setting are conflicting, which is mainly due to differences in study design and size of patient population analyzed, differences in the definition of low and high risk patients, and differences in clinical endpoints. Whereas most studies demonstrated that G-CSF significantly shortens the time of neutropenia, the effect of the administration of the costly hematopoietic growth factor on the incidence of febrile neutropenia, decrease in antiobiotic usage, decrease in hospitalization, influence in the rate of complete remission, and, most importantly, the improvement of disease-free survival are less clear and depend on a number of clinical factors. Systematic review of the literature suggested that the prophylactic use of G-CSF should be reserved for patients with a high incidence of febrile neutropenia, e.g., patients with an expected incidence of fever and neutropenia 40%, which is the fact for children with high-risk ALL or B-cell NHL. 6 In contrast to patients with lymphoblastic leukemia and solid tumors, there was much concern about the use of G-CSF in patients with AML, since most of myeloid leukemia cells express receptors for the growth factor, and G-CSF has been shown to induce proliferation of leukemic blasts in vitro.7 However, clinical studies in adult patients have not shown harm from growth factor administration when given after completion of induction chemotherapy for AML. In addition, most studies demonstrated that the addition of G-CSF resulted in a decreased time of neutrophil recovery and in a reduction of duration of hospitalization and antibiotic use. The administration of G-CSF, however, did not improve complete remission rate, and except for two smaller studies, had no effect on patient survival. Therefore, in the recommendations by the American Society of Clinical Oncology, it is stated that G-CSF use can be considered in this setting if benefits in terms of possible shortening of hospitalization outweigh the costs of G-CSF use. 6 In contrast to adult patients, there are limited data in the pediatric population regarding the use of G-CSF in patients with AML. One non-randomized study, which compared two consecutive pediatric patient populations with AML treated according to the clinical trial CCG-2891, showed that the administration of G-CSF significantly shortened the time of neutropenia and also reduced the hospital stay.8 In contrast, no effect of the growth factor on the incidence of grades 3 and 4 toxicities, on induction remission rates and overall-survival and event-free survival was seen.

The recently closed multicenter clinical trial AML-BFM 98 prospectively evaluated the impact of G-CSF on hematopoetic recovery, infectious complications and five-year event-free survival in pediatric patients with AML. Patients were randomized to receive or not to receive G-CSF (5 g/kg/day) after induction 1 (cytarabine, idarubicin, etoposide; AIE) and induction 2 (high-dose cytarabine and mitoxantrone; HAM). Patients with FAB M3 subtype and patients who had more than 5% blasts in the bone marrow on day 15 were excluded from randomization. Hundred-fifty-eight children were randomized to receive G-CSF, and 169 patients not to receive G-CSF. The G-CSF group had a significantly shorter duration of neutropenia both after induction 1 and after induction 2, but the incidence of fever without identifiable source during neutropenia was reduced only after induction 2 (P = .03). In contrast, no impact of G-CSF was found on the incidence of grade 3 and 4 infections (fever due to an identified pathogen/sepsis syndrome) and mucositis grades 3 and 4. Five-year event-free survival did not significantly differ between the groups with and without G-CSF (P = .63). Whereas no difference of the rate of relapses was seen in the overall group and in high-risk patients, there was a small but statistical significant higher rate of relapses in standard-risk patients who received G-CSF compared to standard-risk patients who did not receive the hematopoietic growth factor (P = .043). To date, one can only speculate on the explanation of this surprising observation, which might be due, at least in part, to different numbers of G-CSF receptors on the leukemic blasts or to a different sensitivity of these receptors, which is currently under investigation.

In conclusion, the administration of G-CSF to adult patients with AML seems to be safe, but whether the clinical benefit outweighs the costs of the growth factor has to be determined. In contrast, the use of G-CSF in children with AML cannot generally be recommended until it becomes clear whether there is a subgroup of patients in which the administration of the growth factor can increase the risk of relapse. 

The introduction of the hybridoma technique by Kö hler and Milstein in 1975 and the development of genetic engineering to convert existing mouse monoclonal antibodies (mAbs) into mouse-human chimerized Abs or humanized reagents have been central to the clinical use of therapeutic mAbs. Much hopes have been raised as to the therapeutic potential of mAbs as magic bullets in the field of hematologic malignancies and solid tumors [1] . However, clinical progress in this field has been frustratingly slow, and only by 1997 the first unconjugated mAb (rituximab) has been approved for the treatment of relapsed low-grade follicular non-Hodgkin's lymphoma (NHL) [2] . During the last eight years, nine anti-cancer mAbs have been approved for human treatment by the registration authorities and many other molecules, partly with novel effector functions, against a variety of targets are in pre-clinical development or in early stages of clinical evaluation [3] . This breakthrough is mainly due to the availability of recombinant engineering technologies that allow the production of chimeric or humanized reagents, with long half-lives, reduced chance of stimulating antiantibody responses, and efficient interaction with natural effectors [4] . Recent surveys suggested that nowadays over a quarter of all biotech drugs in development are mAbs, and, in addition to reagents against cancer, several other mAbs with novel therapeutic applications, including inhibition of alloimmune and autoimmune reactivity, antiplatelet therapy, and antiviral therapy have obtained marketing approval by the registration authorities in the USA (i.e., FDA) and Europe (i.e., EMEA) [1] . Despite these technical developments and promising clinical results in phase II/III studies, there are still many open questions, e.g., as to the mechanisms of action of and resistance to therapeutic mAbs, and the costeffectiveness as well as quality-of-life benefits in specific tumor entities. Furthermore, clinical studies on the long-term safety of mAbs are urgently needed.

Here, advances in the current understanding of biological responses to mAbs employed for the treatment of hematologic malignancies will be briefly reviewed. In addition, recent data from clinical trials or case reports regarding short-time adverse drug reactions (ADRs) and long-term safety of these mAbs are described. Current clinical results with mAbs in hematologic malignancies are reviewed elsewhere in detail [5] [6] [7] [8] [9] .

The determinants of the biological activity of a monoclonal antibody depend on the nature of the target antigen, the specificity of the mAb, the antibody effector mechanisms and the development of resistance mechanisms by the tumor cell or by the host [10] . An optimal target for mAb therapy requires an antigen highly expressed on the cellular surface of the tumor cell without any free antigen in the plasma. The expression of the target antigen should not be downregulated but should remain at high levels, even after the mAb has bound to the antigen. The specificity of the mAb is not only mandatory for an effective binding of the target antigen, but also influences to a great extent the effector mechanisms like complement dependent cytolysis (CDC) and antibody dependent cellular cytotoxicity (ADCC).

The target antigens: CD20, CD52 and CD33 CD20 is an integral transmembrane nonglycosylated hydrophobic phosphoprotein present on the surface of normal precursors and mature Bcells. The precise function is still unknown, but it seems to play an important role in B-cell differentiation, activation and growth [11] . CD20 remains expressed throughout B-cell ontogeny until terminal differentiation to plasma cells. Expression of CD20 is B-lineage restricted and is present on about 90% of all B-cell NHLs and in about 50% of precursor B-cell lymphoblastic leukemia [8] . The antigen displays optimal features for mAb therapy since it is stable in the membrane of B-cells and it is not shedded, modulated or internalized in response to antibody binding [12] .

CD52 antigen is a glycoprotein with a short peptide sequence of 12 amino acids and a large complex N-linked oligosaccharide that is attached to the membrane by a glycophosphatidylinositol (GPI)-anchoring structure [13] [14] [15] . The molecule is constitutively expressed in membrane lipid rafts found in normal and neoplastic lymphocytes, monocytes, eosinophiles, macrophages and parts of the male reproductive system. It is not found on erythrocytes or stem cells. On a large proportion of lymphoid cell malignancies CD52 is expressed in high density. The function of CD52 remains unknown.

CD33 is a sialic acid-dependent cell adhesion molecule and belongs to the immunoglobulin superfamily subset of sialic acid binding immunoglobulinrelated lectins. The biological function and the natural ligand of CD33 are unknown. The cytoplasmic tail has two tyrosine residues that closely resemble immunoreceptor tyrosine-based inhibitory motifs (ITIMs). Upon receptor crosslinking or pharmacological treatment (e.g. pervanadate), these tyrosines are phosphorylated and provide docking sites for recruitment of and activation of Src homology-2 (SH-2) domain containing tyrosine phosphatases (SHP-1 and SHP-2). It is not known whether CD33 engagement by gemtuzumab activates similar signalling or whether these events affect internalization and intracellular trafficking of the anti-CD33 immunoconjugate [9] .

The antibodies: Rituximab, ibrutumomab tiuxetan, alemtuzumab and gemtuzumab ozogamicin Rituximab (IDEC-C2B8) is a chimeric mouse/human monoclonal antibody (mAb), consisting of a glycosylated IgG1 kappa immunoglobulin with murine light-and heavy-chain variable regions (Fab domain) and human kappa and [16, 17] . A number of in vitro studies were performed to confirm the specificity and affinity for the CD20 epitope. The apparent binding affinity constant was 5.2 x 10-9M. The binding of human complement C1q and complement dependent cytotoxicity and antibody dependent cytotoxicity was documented by fluorescein conjugation and 51Cr release. The tissue specificity was demonstrated in several human cross reactivity studies. The antibody, ibritumomab is a 1316 amino acid murine IgG1 antibody consisting of two light chains of 213 residues and two heavy chains of 445 residues. The antibody contains the entire murine light and heavy chain variable regions and the murine gamma 1 heavy chain and kappa light chain constant regions. The molecular weight calculated from the primary sequence of the reduced, non glycosylated form is 144 248 Daltons. Ibritumomab tiuxetan (IDEC-2B8-MX-DTPA) is defined as the active substance in Zevalin. It is obtained by chemically linking the monoclonal antibody ibritumomab (IDEC-2B8) to the amino-directed bifunctional chelate MX-DTPA (tiuxetan), a derivative of diethylenetriaminepentaacetic acid (DTPA) [18] . Ibritumomab tiuxetan achieves selective targeting CD20+ cells, which are inherently sensitive to radiation. The radionucleide Yttrium-90 (half-life of 64 hours) emits pure highenergy beta radiation with a local tissue penetration (5 to 10 mm) and effect.

The alemtuzumab molecule is a genetically engineered human IgG1 kappa mAb with a molecular weight of approximately 150 kD [13, 19] . The humanised antibody was made by the insertion of six complementarity-determining regions (CDRs) from an IgG2a rat monoclonal antibody into a human IgG1 immunoglobulin molecule. The alemtuzumab molecule consists of two light chains of~24 kD (214 amino acids) and two heavy chains (450 amino acids) linked together by two intra (light chain -heavy chain) disulphide bridges and two inter (heavy chain -heavy chain) disulphide bridges to form the typical Yshaped IgG1 molecule. Each molecule also contains a total of 12 intra-chain disulphide bridges and an asparagine residue (301) in each heavy chain, which is glycosylated.

Gemtuzumab ozogamicin consists of a humanized IgG4 anti-CD33 monoclonal antibody joined to N-acetyl-g-calicheamicin dimethyl hydrazide (CalichDMH) [9] .

Effector mechanisms of most unconjugated mAbs are immune-mediated and consist mainly of complement-dependent cytolysis (CDC) and antibodydependent cellular cytotoxicity (ADCC) leading to cell lysis or they directly influence receptor-based inhibiting or activating signalling pathways within the target cell leading to either a block of proliferation and/or apoptosis [12] .

Conjugated mAbs exploit different pathways of destruction than given by the innate immune system. In the case of ibritumomab tiuxetan the addition of the radioisotope 90Yttrium improves the activity of the mAb through targeted radiation, whereas the gemtuzumab ozogamicin conjugate takes advantage of the trojan horse principle by rapid internalization and translocation of the cytotoxic calicheamicin moiety into intracellular and intranuclear compartments of the cell [9, 12, 20] .

Most of the approved therapeutic mAbs are of the IgG1 subtype (Table 1) [19] [20] [21] . They can activate the complement cascade with subsequent deposition of complement components onto the target cell membrane and cell lysis. Binding of the IgG antibodies to the target antigen leads to conformational changes which allow the binding of the C1q molecule to the Fc-portion of the antibody, thus initiating the classical pathway of the complement cascade. A prerequisite for the activation of C1q is the aggregation of IgG molecules on the cell membran, so that a single C1q molecule binds two or more immunoglobulins. This is followed by a complex sequence of enzymatic events leading to the formation of the membrane attack complex (MAC) consisting of the complement factors C5b, C6, C7, C8. The addition of C9 creates the MAC which results in a large pore that spans the membrane of the cell being attacked, allowing ions to flow freely between the extracellular and intracellular spaces causing water to flood into the cell and ultimately burst the cell from osmotic pressure.

Both, rituximab and alemtuzumab exert CDC as one mechanism of action. It has been shown that rituximab-mediated CDC depends primarily on CD20 protein expression [10, 21, 22] . In cells with higher CD20 expression CDC is more rapidly and efficiently triggered by rituximab than in cells with low CD20 expression. This is reflected by the lower rate of response to rituximab in B-CLL due to the low CD20 expression compared to other B-lymphoproliferative disorders with higher CD20 expression. An important mechanistic observation involves the ability of rituximab to translocate CD20 into lipid rafts, thereby favoring the activation of lytic complement leading to a more rapid and efficient cytolysis [23] . Additional support for the role of complement activation in rituximab-induced cytotoxicity includes the observation that complement is consumed during rituximab therapy. Moreover, blocking of CD59 and/or CD55 function with specific antibodies significantly increased CDC in cells treated with rituximab.

In vitro studies of cytotoxic effects of ibritumomab and for the IgG4 component of gemtuzumab could not demonstrate any ADCC or CDC for these mAbs [9, 20] .

The major effector mechanism of rituximab and alemtuzumab is given by ADCC mediated through ligation of the Fc portion of IgG1 or IgG3 mAbs to Fc receptors (FcRI, FcRII, FcRIII ) expressed by accessory cells like macrophages, NK-cells and granulocytes [3, 10] . Binding of NK cells to the target leads to subsequent activation of the NK cell with release of cytoplasmic granules containing perforin and granzymes. Perforin attacks the target cell in a similar way like the MAC by forming pores into the membrane, whereas the granzymes are serine proteases that activate apoptotic cascades through caspase cleavage resulting in DNA degradation. Thus, the release of the cytoplasmic granules at the cellular contact point results in the rapid destruction of the antibody-coated target cell [3, 10] . Other effector cells bearing Fc receptors like granulocytes or macrophages can also participate in tumor killing mainly by phagocytosis, but NK cells are thought to be the main effector cells of ADCC.

Induction of apoptosis is a significant effector mechanism for rituximab and alemtuzumab [10, 12] . Apoptosis is initiated after cross-linking of the target antigen CD20 or CD52 by the respective mAb. This has been observed for CD20 and CD52 in malignant human B-and T-cell lymphoma cell lines [22, 24, 25] . Although, the degree of apoptosis has been highly variable, crosslinking of membrane bound target antigens seems to be critical for apoptosis to occur [12] . The aforementioned lipid rafts appear to play an important part not only for CDC but also for CD20-induced caspase activation by increased calcium influx and induction of close proximity with other raft localizing molecules, including scr-family kinases [26] . The role of numerous other signaling pathways that have been invoked in rituximab-induced apoptosis like STAT3, ERK1/2 and NF-kB remains to be determined [27] .

Apoptosis could not be induced by ibritumomab in its monomeric form, but only after hyper-crosslinking via secondary antibodies [20] .

Resistance can be due to tumor-or host-associated features that interfere with the effector mechanisms of the mAb. Since most of the numerous effector mechanisms of mAbs are not completely understood, the nature of both primary and secondary tumor associated resistance is still elusive and requires further investigations [12] .

In an effort to predict sensitivity of subsequent rituximab therapy the gene expression patterns of primary lymphoma specimens were studied [28] . Many of the high expressing genes in the non-responder group were involved in cellular immune response, suggesting that the microenvironment, more than the malignant cell itself, may have a profound impact on responsiveness to rituximab [28] . These data were in line with a larger gene expression profiling study that correlated survival of patients with follicular lymphoma with molecular features of tumour infiltrating immune cells [29] .

Host-associated resistance mechanisms are mainly due to functional polymorphisms of the Fc-receptor genes for CD16(FcRI), CD32(FcRII) and CD64(FcRIII). The polymorphism that encodes FcRIIIa with either a phenylalanine (F) or a valine (V) at amino acid position 158 results in a higher affinity for human immunoglobulin and increased ADCC, when V is the allotype. This has been shown to be of clinical significance for patients with follicular lymphoma treated with rituximab in a study by Ghielmini et al. [30] . The objective response rate at 12 month was 90% in FCGR3A-158V homozygous patients compared with 51% in FCGR3A-158F carriers [30] .

Since alemtuzumab recruites essentially the same effector mechanisms of the innate immune system like rituximab (CDC and ADCC) it underlies similar hostassociated resistance mechanisms. However, this has not been studied in such detail as for rituximab. Nonetheless, there are some specific features due to the GPI-anchored CD52 [10] . Mutations in the PIG-A gene can lead to CD52 deficiency of T-and B-cells closely resembling the phenotype of lymphocytes from paroxysmal nocturnal hemoglobinuria (PNH). The emergence of CD52 deficient lymphocytes after treatment with alemtuzumab has been described as well as B-CLL patients with constitutive PIG-A mutations present at low levels before treatment resulting in the emergence of a PNH-like clone after treatment [31, 32] .

Potential mechanisms of resistance to gemtuzumab include the escape of leukemic cells due to CD33-negativity, or resting of CD33-positive cells in the G0-phase of the cell cycle [33] . Of clinical relevance is the multiple drug resistance of the leukemic clone by permeability glycoprotein (Pgp)-or multidrug resistance protein 1 (MRP1) -mediated drug efflux [34] . Alterations in intracellular signaling or cell death pathways and antiapoptotic effects of Bcl-2 or Bcl-Xl activity have been reported recently to interfere with the response of human acute myeloid leukemia cells to gemtuzumab ozogamicin [35] . Furthermore, high circulating CD33 antigen burden can compromise drug delivery to the bone marrow and limit the efficacy of the anti-CD33 treatment [36] .

Therapeutic infusions of mAbs are typically associated with characteristic toxicity syndromes that have been related to the following mechanisms: activation of inflammatory cells or mediators after binding of the mAb to its target (e.g., cytokine-release syndrome); rapid destruction of predominantly malignant cells leading to electrolyte abnormalities and metabolic derangements (i.e., tumor lysis syndrome); suppression of physiological functions in line with the specificity of the mAb (e.g., infectious complications); and the xenogenic nature of the mAb used, especially when the mAb is administered without associated immunsuppression (i.e., formation of human anti-murine, HAMA, or antichimeric antibodies, HACA) [1, 2, 37] . The most common short-term ADRs observed after the administration of mAbs are infusion-related and usually occur during or within 24 hours of the first infusion of the mAb [37] . These infusionrelated side-effects are mediated by inflammatory cytokines such as TNF-, IFNinterleukin(IL)-6, and IL-8, and consist of chills, fever, headache, asthenia, dizziness, pruritus, urticaria, angioedema, myalgia, arthralgia, nausea, vomiting, diarrhea, bronchospasm, dyspnea, vasodilation, hypotension, and arrhythmias. Occasionally, severe manifestations including pulmonary infiltrates, acute respiratory distress syndrome (ARDS), myocardial infarction, ventricular fibrillation, and cardiogenic shock have been reported [2, 19] . Postmarketing safety experience indicates that infusion-related adverse reactions after rituximab or alemtuzumab have resulted in death in only a very small proportion of patients (i.e., 0.04-0.07%) [2, 19, 21] . The cytokine-release syndrome is more frequently observed in patients with higher numbers of tumor cells in their peripheral blood. Management of infusion-related adverse effects include symptomatic treatment with antipyretics, antihistamines, intravenous fluids, and, if necessary, corticosteroids. For more severe symptoms, treatment includes temporary discontinuation of mAb infusion and reintroduction at a lower rate 30 to 45 minutes later.

Hypersensitivity reactions, including anaphylaxis, have occasionally been reported following the use of mAbs in hematologic malignancies, and clinical manifestations of hypersensitivity reaction may be similar to those of cytokinerelease syndrome. In contrast to cytokine-release syndrome, however, clinical manifestations of true hypersensitivity reaction typically occur within minutes of starting the mAb infusion [2] .

Tumor lysis syndrome, though rarely occurring in hematologic malignancies secondary to a rapid decrease in tumor volume after infusion of mAbs, has been reported with the administration of rituximab, alemtuzumab, and gemtuzumab [37] . Tumor lysis syndrome is usually characterized by hyperuricemia, hyperkalemia, hyperphosphatemia, hypocalcemia, and an increase in lactate dehydrogenase. Renal failure, sometimes requiring dialysis, has been associated with the use of rituximab, alemtuzumab, and gemtuzumab in NHLs and acute myeloid leukemias, and fatal outcomes resulting from tumor lysis syndrome have been reported. Patients with a high tumour burden and greater than 25 x 109/l circulating malignant cells are at increased risk. Tumour lysis syndrome may be prevented by leukoreduction (e.g., by cytotoxic drugs), hydration, and allopurinol prior to administration of mAbs [37] .

Rituximab and ibritumomab efficiently kill both malignant and non-malignant CD20-positive cells, and normal B-cells are rapidly depleted following administration of these mAbs [2] . For example, four infusions of rituxumab at a conventional dose typically deplete B-cells for 2-6 months, with levels returning to normal by 12 months [2] . B-cell depletion after rituximab was associated with decreased serum immunoglobulins only in a minority of patients. Pooled data from 356 patients receiving rituximab monotherapy showed an overall incidence of 30% for infectious diseases, with severe infectious events, such as sepsis, occurring in 1-2% of patients [21, 37] . Recently, unusual viral infections (progressive multifocal leukoencephalopathy and cytomegalovirus disease) have been reported in four patients after highdose chemotherapy with autologus blood stem cell rescue and peritransplantation rituximab [38] . These cases as well as rare reports on pure red cell aplasia due to parvovirus infection following treatment with polychemotherapy and rituximab for B-cell lymphoma highlight the need to be vigilant for infections caused by delayed immune reconstitution after treatment with rituximab or other mAbs [39] . Reactivation of hepatitis B virus (HBV) infection despite the presence of anti-HBs, resulting in fulminant hepatitis, hepatic failure, and death, have been reported in patients treated with rituximab as monotherapy or in combination with cytotoxic or immunomodulatory drugs (e. g., IFN-alfa-2b) [37, 40, 41] . Therefore, the cautious use of rituximab and ibritumomab in patients with anti-HBs who have serologic evidence of previous HBV infection has been recommended [40] . In addition, patients who are still at risk for reactivation of latent HBV infection or with hepatitis C virus during or after rituximab (with chemotherapy) should be considered for prophylaxis with lamivudine [41] . Due to its ability to completely deplete CD52+ cells, including B and T-lymphocytes, monocytes, and NK cells from the peripheral blood, serious, sometimes fatal, opportunistic infections (bacterial, viral and fungal) have frequently been reported with alemtuzumab therapy. In clinical trials without anti-infective prophylaxis, 66% of patients experienced at least one infection [19] . Therefore, prophylactic therapy against PCP pneumonia and herpes viral infections is recommended upon initiation of therapy with alemtuzumab and for at least 2 months following last dose or until CD4+ counts 0.2 x 109/l. Due to the immunosuppressive effects of rituximab, ibritumomab, and alemtuzumab, patients who have recently received these mAbs should not be immunized with live viral vaccines [37] .

Evidence of HAMA or HACA were observed in a low proportion of patients treated with rituximab and ibritumomab (0,4-1,5%) and humoral responses to the calicheamicin-linker complex, but not to the mAb of gemtuzumab, were demonstrated in 2 of 40 AML patients [37] . No severe clinical consequences of antibody formation have yet been reported. However, occasional cases of serum sickness in association with rituximab have been reported, and clinicians should be aware that it can occur [2] . Myelosuppression with grade 3 or 4 neutropenia, thrombocytopenia, or anemia occur frequently with alemtuzumab, gemtuzumab, and ibritumomab [37] . Although pluripotent hematopoietic stem cells are CD33-and CD52negative, more differentiated progenitor cells express these antigens, and are therefore targeted by gemtuzumab or alemtuzumab which may result in severe hematologic toxicity. Myelosuppression after treatment with ibritumomab has correlated with pre-treatment tumor marrow involvement and platelet counts and is due to beta-emission by Yttrium-90 that induces cellular damage, e.g., of hematopoietic progenitor cells, through the formation of free radicals [37] . Outside of clinical trials, ibritumomab should currently not be administered to patients with > 25% lymphoma involvement in their marrow or impaired bone marrow reserves, as indicated, e.g., by prior myeloablative therapy, platelet counts < 100 x 109/l and hypocellular bone marrow [37] . Despite its favourable hematological profile with grade 3 or 4 hematologic toxicity occurring in only 2-6% of patients receiving rituximab alone, data from clinical trials indicate that protocol adjustments and dose reductions are needed to decrease the hematologic toxicity when rituximab is administered in combination with fludarabine [2] . Interestingly, several case series have recently reported on delayed-onset neutropenia after administration of rituximab that may persist for between several days and several months, before undergoing spontaneous recovery [2, 42] . In most patients, the features were suggestive of immunemediated neutropenia. Several mechanisms have been suggested to explain delayed-onset neutropenia, including an expansion of T-large granular lymphocytes in the bone marrow, a correlation between certain genotypes of polymorphism at the IgG Fc receptor and neutropenia following rituximab therapy, the transient production of autoantibodies during the recovery of normal B lymphocytes, and deranged cytokine production due to B-cell depletion [2, 42] . A direct toxic effect of rituximab is unlikely, since hematopoietic progenitor cells and granulocytes do not express CD20.

Several cases of secondary malignancies, especially myelodysplastic syndrome (MDS) and AML, have been reported after treatment with ibritumomab, suggesting a possible link between radioimmunotherapy and secondary MDS or AML [37] . Thorough assessment of the incidence of MDS and AML, particularly with more patients and a longer follow-up period, is therefore needed.

Serious or potentially fatal ADRs are often detected after cancer drugs, including mAbs, are widely used in oncology practice [43] . Licensing clinical trials are designed primarily to identify benefits and common side effects. However, the size of these premarketing clinical trials is often too small (ranging from 100 to usually less than 1000 patients) to identify rare but potentially serious ADRs [44] . First results from the recently initiated Research on Adverse Drug events And Reports (RADAR) project in the postmarketing setting have demonstrated serious ADRs of mAbs administered in hematologic malignancies which occur with an incidence rate from 1 in 170-200 patients (rituximab: severe infusion reactions) up to 1 in 3-7 patients (gemtuzumab: sinusoidal obstruction syndrome) [43, 44] . These findings underscore the need for continued vigilance and better postmarketing surveillance, especially for new drugs such as mAbs passing through fast track or accelerated approval processes, in order to improve patient safety. In the last decade, inhibition of signal transduction pathways by small molecule inhibitors has emerged as a very promising approach for the treatment of solid and hematopoietic malignancies. This inhibition might either be directed against the activity of the transmembrane or cytoplasmic receptor itself or against signalling molecules that act downstream in response to receptor stimulation and regulate growth and apoptosis. Products of K-RAS, N-RAS and H-RAS are small-molecule membranebound guanine nucleotide binding proteins that play a crucial role in a number of signal transduction pathways and regulate many cellular functions such as proliferation and differentiation. Inhibition of RAS family proteins represents a very promising therapeutic approach since members of this family of small GTP hydrolases might be either activated by stimulated or mutated tyrosine kinases or carry mutations in hot spot regions themselves.1,2 RAS mutations usually detected in patients with a variety of different malignancies occur at codons 12, 13 or 61. These positions are critical for regulation of RAS activity and also affected in experimentally induced tumors of mice or rats. 3 The mutated RAS is still able to form complexes with GTPase activating proteins, but has lost its ability to hydrolyse bound GTP. That leads to spontaneous activation of downstream effectors such as RAF and PI-3-kinase. In cell culture models, activating RAS mutations induce loss of contact inhibition, substrateindependent growth and enhanced DNA synthesis rate. In adult de novo AML, activating point mutations of N-RAS have been detected in 20% to 30%, so that farnesyltransferase inhibitors that directly inhibit RAS function by disturbing its proper posttranslational processing and membrane binding are subject of current clinical testing. The non-peptidomimetic competitive farnesyltransferase inhibitor R115777 (tipifarnib, Zarnestra) has been studied across a wide range of different malignancies, e.g. in patients with solid tumors or hematopoietic neoplasia of myeloid origin (myelodysplastic syndrome and refractory or relapsed acute myeloid leukemia). Dose-limiting side effect of this compound consists of myelosuppression that resembles to that seen with conventional chemotherapy.4 Intriguingly, not only patients with RAS mutations respond to treatment with R115777, but also patients without so that R115777 might interfere with targets apart from RAS.

Another possible therapeutic interference might be the inhibition of signalling molecules downstream to RAS, e.g. MEK-1. Compounds like PD98059 have been well characterized in vitro as well in vivo. Due to its mechanism of action the activity of PD98059 is merely restricted to the inhibition of the ERK pathway.

As mentioned above, constitutive activation of RAS is not only caused by point mutations, but indirectly as well by permanent activation of upstream receptors or negative regulators such as neurofibromatosis NF-1 protein. 5 For RAS activity being dependent on GTPase activating proteins accelerating the rate of GTP hydrolysis, an association of loss-of-function mutations e.g. in NF-1 protein with development of myeloid leukemias has been shown.

The receptor tyrosine kinase FLT3 that has been shown to activate the RAS signalling cascade plays a crucial role in the proliferation and survival of multipotent progenitor cells. Primary AML blasts express FLT3 and stimulation with FLT3 ligand leads to proliferation and resistance to apoptosis by induction of BCL-2. Activating mutations of the juxtamembrane and tyrosine kinase domain of FLT3 represent the most frequent genetic alterations in acute myeloid leukemia. They can be detected in 30-35% of all patients. 6 The presence of FLT3 length mutations (FLT3-LM) has been associated with an impaired clinical outcome, i.e. increased relapse rate and impaired overall survival, in all cytogenetically defined AML risk groups. The overexpression of FLT3 ligand leads to a predisposition for leukemia in a murine bone marrow transplantation model. The FLT3 receptor represents an attractive molecular target in AML due to its essential pro-proliferative and anti-apoptotic activity. In the last years, a number of specific small molecule inhibitors have been identified that selectively target FLT3. These inhibitors abolish the autophosphorylation of mutated FLT3 receptor and induce growth arrest and apoptosis in FLT3 transformed AML cell lines. A number of specific FLT3 inhibitors are currently in clinical phase I/II studies, e.g. CEP-701, MLN-518 and PKC412. These substances prolong the latency time of the FLT3 induced myeloproliferative syndrome in mouse models.

In clinical studies all substances are well tolerated and lead to reduction of leukemic cell mass as well as partial remissions in extensively pre-treated AML patients. In contrast to chronic myeloid leukemia (CML) in chronic phase, AML represents an aggressive and genetically very heterogeneous disease. The selection of inhibitor resistant subclones is facilitated by the high proliferation rate and apoptotic resistance of AML blasts. Moreover, it is substantiated by experimental data that FLT3 mutations require additional genetic events for induction of a leukemic phenotype. Thus, a rational and clinically orientated treatment of AML will require a combination therapy in contrast to therapy of CML in chronic phase.

Apart from FLT3, a variety of other receptor tyrosine kinases is found to be mutated in solid and hematopoietic malignancies. PTK inhibitors that target epidermal growth factor receptor (EGFR) have been approved for the indication in Europe and the US. EGFR is overexpressed and mutated in patients with non-small cell lung cancer so that it has been recognised as target of increasing importance. Clinical phase I and II studies have demonstrated a distinct response of 10-20% of patients.

The role of receptor tyrosine kinases is not only dependent on their activation and dimerization by ligand binding but likewise on the dynamic equilibrium between (auto) phosphorylation and dephosphorylation. Activated phosphotyrosine kinases present high affinity binding sites for negative regulators like tyrosine phosphates SOCS and SHP-2. Missense mutations in PTPN11, the gene encoding for SHP-2, have been reported in a small percentage of patients with myelodysplastic syndrome (MDS), acute myeloid leukemia (AML), and acute lymphoid leukemia. Three somatic point mutations of SHP-2 have been shown to induce macrophage progenitor hyperproliferation in response to GM-CSF but not to M-CSF.7 Small molecule inhibitors targeting SHP-2 tyrosine phosphate have been reported,8 but are currently not in clinical testing.

Contrary to receptor tyrosine kinases, cytokine receptors do not contain an innate kinase domain. The activation of this receptor class is dependent on the physical association with non-receptor kinases, e.g. JAK2. Very recently, a recurrent gain-of-function mutation of JAK2 has been identified (JAK2-V617F) that seems to influence the pathogenesis of myeloproliferative syndromes, i.e. polycythemia vera, essential thrombocythemia and myeloid metaplasia with myelofibrosis. 9 The V617F point mutation resides in the auto-inhibitory JH2 domain and leads to constitutive phosphorylation and hypersensitivity of EPO receptor that exerts its signal transduction via the JAK-STAT pathway. Thus, therapeutic inhibition of mutated JAK might also represent a valuable target for small molecule inhibitors.

Last year we reported interim results of a randomised phase III trial in patients with relapsed/refractory stage III/IV positive follicular NHL, demonstrating that the addition of rituximab (R) to CHOP chemotherapy for remission induction as well as rituximab maintenance treatment significantly improves the clinical outcome (Blood 2004;104:169a). Based on these interim results, randomisation to the induction part of the trial was halted and the protocol was amended.

Patients with stages III or IV follicular lymphoma at initial diagnosis and relapsed after or resistant to a maximum of two non-anthracycline containing systemic chemotherapy regimens, were randomized to remission induction with either 6 cycles of standard CHOP (once every 3 weeks) or CHOP + R (375 mg /m2 at day 1 of each cycle of CHOP). Those with a complete or partial remission after 6 cycles of therapy underwent a second randomization to no further treatment (observation) or maintenance treatment with R (375 mg/m2 once every 3 months) until relapse or for a maximum period of two years.

At the time of the preplanned second interim analysis (February 2004) 461 patients had been included. Of these, 369 could be evaluated for response (188 CHOP; 181 R-CHOP). Both treatment arms yielded similar partial response rates (CHOP: 53.7% -R-CHOP: 52.5%), but highly significant different CR rates (CHOP: 18.1%; R-CHOP: 30.4%; p=0.0004). Of 319 patients randomized for maintenance treatment, 268 were evaluable (132 observation; 136 R maintenance). A highly significant advantage was observed in progression free survival in patients randomized to R maintenance, when compared with the observation arm (median PFS 38 vs.15 months; p<0.0001). At that time there was no impact on OS: the observed difference (in favor of R maintenance) was not significant for an interim analysis. Toxicity of CHOP and R-CHOP induction was similar, and R maintenance was associated with minimal toxicity. These results of the planned second interim analysis showed that the formal criteria for stopping the trial had been met. It was concluded that this is the first trial to show that: 1) in patients with relapsed/resistant follicular lymphoma R-CHOP remission induction results in a highly significant increase in CR rate as compared to CHOP; 2) rituximab maintenance treatment significantly improves PFS in patients responding to induction treatment. The final analysis of the study results will be performed in September 2005, with an additional follow-up of at least 18 months. The final results will be presented at the meeting.

The concept that only a subpopulation of rare cancer stem cells (CSC) is responsible for maintenance of the neoplasm emerged nearly 50 years ago, however conclusive proof for the existence of a CSC was only obtained relatively recently. The evidence for the existence of CSC was first derived from the study of human acute myeloid leukemia largely because of the availability of quantitative stem cell assays for the leukemic stem cell (LSC). These studies showed that only rare cells within the leukemic clone had the capacity to initiate AML growth following transplant into NOD/SCID mice establishing the hierarchical organization of AML. Recent clonal tracking studies showed that the LSC compartment is composed of different classes of LSC that can be distinguished on the basis of self renewal potential. These findings have important implications for our understanding of the leukemogenic process as well as the design of more effective therapies that eliminate AML based on eradication of the LSC. Interestingly, CSC were recently proven to be present in breast cancer and brain cancer demonstrating the universality of this principle for both solid and hematopoietic tumors. Much work needs to be done to characterize CSC from all types of cancer and to use this information to devise CSC-targeted therapies.

A fundamental problem in cancer research is identification of the cell type capable of initiating and sustaining growth of the neoplastic clone in vivo. The key to solving this riddle lies in determining whether every cell within the neoplasm has tumor-initiating capacity, or whether only a rare subset of cellsso-called "cancer stem cells" (CSC) -is responsible for maintenance of the neoplasm [15] . The existence of CSC was first proposed over 40 years ago, providing an explanation for observed functional heterogeneity within tumors (reviewed in [15] ). Many studies showed that only a small subset of cancer cells is capable of extensive proliferation in vivo [2] and in vitro [4, 11] , using quantitative assays. However, diametrically opposed stochastic (e.g. tumor is functionally homogeneous since every tumor cell has a low but equal probability of behaving as a CSC) and hierarchy (e.g. tumor heterogeneous and organized as a hierarchy with only a small definable subset of CSC that are able to reproduce and sustain the tumor hierarchy) models were proposed to explain the experimental data (see [15] ). The key advance to resolve this dilemma came from our discovery of AML-LSC as the only cell capable of establishing the AML hierarchy [8] , thereby proving the CSC hierarchy model [1] .

By adapting the available quantitative assays for normal human stem cells, we showed that transplantation of primary AML cells into SCID [8] , or NOD/SCID [1] mice resulted in identification of rare cells, termed SCID leukemia-initiating cells (SL-IC), with the capacity for initiating and sustaining leukemic growth in primary and serially transplanted mice proving that had self renewal and thus were true AML stem cells. SL-IC were exclusively isolated within the CD34 +CD38 cell fraction with few exceptions, regardless of the blast population phenotype. These findings rule out stochastic processes as the biological mechanism underlying tumor heterogeneity in AML, and show that, like the normal hematopoietic system, AML is organized as a hierarchy of distinct, functionally heterogeneous cell classes that are sustained by rare LSC.

Based on the consistency of the CD34+CD38-cell surface phenotype of SL-IC among patients regardless of AML subtype and their similarity to the phenotype of normal SRC, we proposed a model of leukemogenesis similar to McCulloch [9] where the cell of origin is the HSC rather than a committed progenitor [1] . In this model, transforming genetic events only occur in primitive stem cells and it is the impact of these transforming events on the stem cell that govern the leukemic phenotype. The alternate view proposes that lineage committed progenitors are the cell of origin and the leukemic cell properties are determined from the commitment stage of the specific progenitor targeted (reviewed in [15] ).

An intermediate view would be that the cell of origin lies in between these extremes, within multipotent progenitor cells that have lost their self renewal capacity but that have not yet made final commitments to a specific lineage [13] . Strong evidence for the stem cell origin of AML came from our clonal tracking studies that established that normal HSC and LSC both possessed the capacity for self renewal as well as the capacity to regulate self renewal to create similar stem cell hierarchies of short-term (ST) and long term (LT) stem cell classes [5] . However, LSC have a much higher capacity for self renewal, probably due to increased symmetric self renewal cell divisions. Thus, the intrinsic self-renewal capacity, as well as the decline in self-renewal capacity (i.e. regulation of self renewal) due to commitment processes, of HSC targeted by the initial leukemogenic event(s) continue to function in the resultant LSC. Indeed the recent finding that the stem cell-specific gene, Bmi-1, plays a key role in the selfrenewal of both normal and leukemic murine stem cells supports this idea. Additionally, other similarities that link LSC and HSC are they are both quiescent and they express drug resistance transporters. Nevertheless, this evidence is still indirect and because of the importance of this fundamental question, the answer to which will provide insight into the first steps of the neoplastic process, it is essential that direct experimental evidence be gathered.

Attempts to identify the cell of origin for murine leukemia have yielded conflicting results. The fusion oncogene MLL-GAS7 induces mixed-lineage leukemias when expressed in HSC or multipotent progenitors, but not in lineage-restricted progenitors [14] . In contrast, both MOZ-TIF [6] and MLL-ENL [3] leads to initiation of identical AML regardless of the target cell population used. However, much lower numbers of transformed HSC compared to progenitors are required for tumor initiation in vivo, and tumors arising from progenitors are oligoclonal not polyclonal, suggesting that the progenitor populations were not functionally homogeneous nor equally susceptible to transformation. Murine models of CML due to inactivation of JunB or expressing bcr/abl must occur in HSC and not more restricted progenitors in order to induce a transplantable myeloproliferative disorder [6, 12] .

No direct studies have been reported for human hematopoietic cells, however one indirect study was performed. A study of patients with t(8;21) AML demonstrated that despite the presence of leukemia-specific AML1-ETO chimeric transcripts in primitive CD34+Thy-1-CD38-cells from leukemic BM, these cells give rise to normally differentiating multilineage clonogenic progenitors, while more mature CD34+Thy-1-CD38+ cells form exclusively leukemic blast colonies (AML-CFC) [10] . This paper concluded that whereas the initial t(8;21) translocation occurs in a primitive stem cell (the "cell of origin"), subsequent events occur in the committed progenitor pool, giving rise to LSC. However, no in vivo LSC studies were carried out and AML-CFU are not LSC [8] , therefore this study is difficult to interpret.

Leukemic stem cells hold the key to understanding the origin and maintenance of AML. Thus, elucidation of these LSC-specific properties will aid in the development of more effective therapy that can be targeted to the most primitive LSC [7] . For example, Jordan et al have shown that LSC have higher levels of constitutive NF-KB activation compared to normal HSC providing an opportunity to devise selective therapies [7] . The discovery of functional complexity in the LSC compartment has critical implications for the investigation of cancerspecific signaling pathways and the development of stem cell targeted AML therapies. Since cancer pathways may function differently within each LSC subclass compared to the bulk leukemic blasts, differential responses to a given therapy may result. Effective therapy must target the highly self-renewing LT-SL-IC within a functionally heterogeneous SL-IC pool that is responsible for aggressively driving the growth and relapse of AML. Current AML therapies typically target proliferating cells, however SL-IC are quiescent making them poorly responsive to such agents. Future therapies may be more successful if they target the altered self-renewal machinery of the LSC to more specifically eradicate the LSC.

Treatment of Acute Myeloid Leukemia in Older Patients: The EORTC-GIMEMA Experience S. AMADORI 1, R. STASI 2 1Department of Hematology, Tor Vergata University Hospital, Rome; 2Hematology-Oncology Unit, Regina Apostolorum Hospital, Albano Laziale, Italy.

Advances in understanding the pathophysiology of acute myeloid leukemia (AML) have not yet led to major improvements in the long term outcome of elderly individuals with this disease. The European Organisation for Research and Treatment of Cancer and Gruppo Italiano Malattie Ematologiche dell'Adulto (EORTC/GIMEMA) leukemia groups are actively exploring the role of new treatment options for these patients. In this article we review the final results of 3 recent trials. The main objective of AML-13, a phase III study, was to determine the efficacy and toxicity of adding glycosylated recombinant human granulocyte colony-stimulating factor to induction chemotherapy. Other objectives were to assess the role of oral versus infusional consolidation chemotherapy, and to evaluate the feasibility of myeloablative chemotherapy with autologous peripheral blood stem cell support as second consolidation course in patients with a good performance status aged 61-70 years. AML-15A was a phase II trial investigating the sequential combination of gemtuzumab ozogamicin (GO) and conventional chemotherapy for induction of remission in patients aged 61-75 years. Finally, the AML-15B trial investigated the feasibility, toxicity, and antileukemic activity of GO monotherapy for remission induction in older patients who were not considered candidates for conventional chemotherapy.

Acute myeloid leukemia (AML) is tipically a disease of the elderly; it develops in nearly 20 per 100,000 persons/year past the age of 70 years in Western countries, but in only 1 or 2 per 100,000 young adults [1] . In the past couple of decades major improvements have been made in the treatment of younger adults with AML. Conversely, the treatment of older patients remains a considerable therapeutic challenge. Older adults are less able to tolerate intensive chemotherapy regimens, often have pre-existing hematologic disorders, and are more likely to have poor-risk cytogenetic abnormalities or expression of the multidrug resistance phenotype [2] . It is therefore not surprising that before 1980, in a time when the availability of both effective antileukemic agents and supportive care measures was limited, very few of these individuals were considered candidates for intensive chemotherapy. However, a randomized study conducted by the EORTC Leukemia group in the late 1980s showed that immediate intensive chemotherapy was better than best supportive care in the elderly. [3] In fact, those who received immediate chemotherapy had their median survival rate almost doubled (21 vs 11 weeks). Since that pivotal study, the EORTC Leukemia group and the Italian cooperative group GIMEMA have conducted a number of collaborative studies in elderly individuals with non-M3 AML. In this review we will present the results of the latest trials.

EORTC-GIMEMA AML-13 trial Strategies to reduce the toxicity associated with intensive chemotherapy have involved the use of attenuated doses of standard regimens and myeloid growth factors. Although a decrease in early death rate can be achieved through dose reduction, response rates are less favorable due to inadequate antileukemic cytotoxicity. Where intensive treatment is attempted, standard practice is thus remission induction followed by a consolidation phase, the latter intended to eliminate residual leukemia cells.

AML-13 was a randomized, open-label, active-controlled, phase III study carried out in 53 European centers [4] . The main objective of this study was to determine the efficacy and toxicity of adding glycosylated recombinant human granulocyte colony-stimulating factor (G-CSF) to induction chemotherapy. Other objectives were (1) to assess the role of oral mini-ICE as consolidation relative to intravenous idarubicin-cytarabine-etoposide (mini-ICE); (2) to evaluate the feasibility of myeloablative chemotherapy with autologous peripheral blood stem cell (autoPBSC) support as second consolidation course in patients with a good performance status (PS) and age of 70 years or younger. The overall study plan is represented in Figure 1 .

A total of 722 patients with newly diagnosed AML, median age 68 years, were randomized into 4 treatment arms: (A) no G-CSF; (B) G-CSF during chemotherapy; (C) G-CSF after chemotherapy until day 28 or recovery of polymorphonuclear leukocytes; and (D) G-CSF during and after chemotherapy. The complete remission (CR) rate was 48.9% in group A, 52.2% in group B, 48.3% in group C, and 64.4% in group D. Analysis according to the 2x2 factorial design indicated that the CR rate was significantly higher in patients who received G-CSF during chemotherapy (58.3% for groups B + D vs 48.6% for groups A+C; P=.009), whereas no significant difference was observed between groups A + B and C + D (50.6% vs 56.4%, P=.12).

Patients who received G-CSF after chemotherapy had a shorter time to neutrophil recovery (median, 20 vs 25 days; P The frequencies of various WHO grade 3 or grade 4 adverse effects after induction cycles 1 and 2 were similar between the groups except for severe hypotension, which was more frequent in patients who received G-CSF after chemotherapy (4.3% for groups C+D vs 1.2% for groups A+B).

A total of 346 patients who achieved a CR were further randomized to receive either the intravenous (n = 172) or the oral mini-ICE regimen for consolidation (n = 174). Baseline characteristics were evenly matched between treatment groups. Of the 346 patients, 15 were not evaluable: 6 in the intravenous and 9 in the oral arm. Overall, consolidation-1 was given to 331 pts, while 182 received consolidation-2.

Grade 3-4 toxicities in the oral vs the i.v. arm were 9% vs 4% for nausea, 10% vs 2% for vomiting, and 20% vs 26% for infection. Profound neutropenia (0.5x109/l), and 31 vs 45 days (p=0.07) for platelet recovery (>150x109/l).

Sixty-one patients aged 61 to 70 years, in CR after the first course of consolidation and with a WHO PS 0-1, were scheduled for autoPBSC transplantation. Stem cells were harvested by leukapheresis after hematopoietic recovery from consolidation-1. In 7 of these 61 patients, the number of CD34+ circulating cells remained below the threshold for starting the apheretic procedure in spite of G-CSF administration. In the 54 harvested patients, a median of 2 aphereses (range, 1 -5) was performed. There was a median collection of 11.7x108 nucleated cells/kg (range, 2.4 -99.8) containing Figure 1 . AML-13 schema 40.2x104 CFU-GM/kg (range, 0 -786.8), and 5x106 CD34+ cells/kg (range, 0.1 -99.8). 12 patients were considered to have a low CD34+ yield (0.5x109/l was 24 days and to platelets >20x109/l was 23 days following transplantation.

Patients have been followed for a median of 4.7 years after the first randomization and 4.4 years after the second randomization. The median overall survival (OS) for all patients was 9.1 months. There were no significant differences in OS between the 4 arms of first randomization. In particular, the estimated median values (97.5% CI) were 7.9 months (5.8-10.4) in group A, 9.2 months (6.7-12.6) in group B, 8.4 months (6.1-10.9) in group C, and 11.5 months (8.3-14.9) in group D. Likewise, there were no differences in event-free survival (EFS) and disease-free survival (DFS) between the various groups.

With regard to the second randomized question (oral vs i.v. mini-ICE consolidation), the median overall survival (OS) was not significantly different between the two randomization groups: 15.7 months in the oral arm and 17.8 months in the i.v. arm (p=0.19, HR=1.17, 95% CI 0.92-1.50). Again, there were no differences in EFS and DFS between the two treatment arms.

Finally, for the whole group of 61 patients eligible for autoPBSC transplantation, the medians were 0.98 and 1.45 years for disease-free survival (DFS) and overall survival (OS) respectively, and the 3-year rates were 21% and 32% respectively.

In summary, the results of this complex trial have shown that although priming with G-CSF can improve the CR rate, the use of G-CSF during and/or after chemotherapy has no effect on the long-term outcome. They also showed that the oral consolidation regimen resulted in an antileukemic effect not significantly inferior to the intravenous regimen. This regimen was less myelosuppressive and associated with less infectious complications and less hospitalization days. Finally, the real impact of autologous SCT on treatment outcome in elderly patients with AML is limited.

These trials aimed at defining the role of gemtuzumab ozogamicin (GO) in older patients with newly diagnosed primary or secondary AML. GO is an immunoconjugate that has already shown promising activity in relapsed AML [5, 6] . It is composed of a humanized anti-CD33 antibody linked to the potent antitumor antibiotic calicheamicin [7] . This agent provides a novel method of drug delivery using the monoclonal antibody to target CD33+ leukemic cells without many of the systemic toxicities associated with traditional chemotherapeutic agents.

The AML-15A trial included patients 61-75 years of age who had WHO PS of grade 0 to 1 [8] . Patients received frontline treatment with GO 9 mg/m2 infused intravenously on days 1 and 15. Following response assessment to GO, patients were started on conventional chemotherapy consisting of the MICE regimen (mitoxantrone, cytarabine, etoposide). No further treatment was planned for complete responders. Among the 57 evaluable patients, 38 (67%) completed the entire sequential treatment as planned. The overall response rate to the entire induction sequence was 54.4% (31/57), with complete remission (CR) in 35.1% and complete remission with incomplete platelet recovery (CRp) in 19.3%. Rates of failure due to treatment-related mortality or resistant disease were 14.1% (3 toxic deaths during the GO segment, 5 during MICE) and 29.9%, respectively. An initial response to GO was documented in 20 patients (35.1%), with CR in 22.8% and CRp in 12.3%; 6 additional patients entered a partial remission. Frontline GO was associated with modest mucosal and gastrointestinal toxicity, but grade 3-4 pancytopenia was universal and prolonged. In particular, after GO complete responders required a median of 37 and 32 days from the first infusion to recover neutrophil and platelet counts >0.5x109/l and >50x109/l, respectively. Hematopoietic recovery post-MICE was not compromised by previous exposure to GO as indicated by the fact that the number of days to recover neutrophil and platelet counts was fully comparable with those of an historical group of patients treated with MICE alone (median of 24 days for both). Hepatic veno-occlusive disease developed in 3 patients after GO and 2 after MICE, resulting in 4 deaths from liver failure. One-year survival at follow-up was 34%. Twelve patients continue in CR/CRp after a median of 226 days.

Based on pharmacokinetic data indicating a satisfactory degree of saturation of CD33 binding sites when a dose of 6 mg/m2 was infused, we speculate that lower doses of GO may prove as effective and less myelotoxic in these conditions, thus allowing a higher proportion of patients to reach the MICE segment of the induction sequence. Such dose-reduction should also result in a lower incidence of severe clinical hepatotoxicity, a recognized and potentially fatal side-effect associated with the administration of GO at full doses. This issue is now being investigated in the current AML-17 phase III trial, which was designed to prospectively address the comparative benefits of lower doses of GO combined with sequential chemotherapy versus chemotherapy alone in patients 61-75 years old with untreated AML.

The main objective of the AML-15B trial was to determine the feasibility, toxicity, and antileukemic activity of GO monotherapy for remission induction in older patients with previously untreated primary or secondary AML who were not considered candidates for conventional chemotherapy ("frail" patients). Patients with AML were allowed to participate in this study if they had an age >75 years and a WHO PS grade 0-2, or an age between 61 and 75 years and a WHO PS grade 2. GO was administered at the dose of 9mg/m2 as a single 2-h i.v. infusion on days 1 and 15.

Patients who achieved a complete remission (CR/CRp) were to receive consolidation with two additional injections of the immunotoxin at the same dose. The overall CR/CRp rate was 17% (95% CI, 8-32%). The CR/CRp rate in patients 61-75 years old was 33% (6/18), and 5% (1/22) in patients older than 75 years. Induction death occurred in seven patients (17%), all aged above 75 years. Overall survival was significantly longer in patients aged 61-75 years than in older individuals (P=0.05), and in CD33+ cases than in CD33-cases (P=0.05). Myelosuppression was universally seen during GO treatment. Grade 3-4 neutropenia was quite prolonged, with complete responders requiring a median of 28 days (range 16-40 days) from the first infusion to recover neutrophil counts >0.5x109/l. The median time to reach a platelet count >50x109/ l was 23 days. The most common nonhematologic adverse events included febrile neutropenia (52%) and infections (40%).

The results of the AML-15B trials suggest that GO, at the FDA-approved dose/schedule, offers a reasonable option for individuals with an age between 61 and 75 years and a PS grade 2. However, it is too toxic and has a limited role in older (>75 years) individuals with AML. In this group of patients, such treatment does not appear to offer substantial benefits relative to conventional nonintensive approaches. Following these considerations, the EORTC and GIMEMA Leukemia Groups have recently activated a prospective randomized study (AML-19), comparing a lower total dose of GO (9 mg/m2) administered according to a more condensed schedule to standard supportive care as initial therapy both in patients with an age over 75 and a PS 0-2, and in patients with an age of 61-75 years and a PS greater than 2. The results of this trial should clarify whether GO has a role in the management of patients with AML for whom intensive cytotoxic regimens would be considered unsuitable. Unique Issues of AML Although acute myeloid leukemia (AML) accounts for approximately 20%-25% of acute leukemia in children, it remains responsible for more than half of the deaths from leukemia. Risk-adapted therapy has been the cornerstone of treatment decisions in pediatric patients with acute lymphoblastic leukemia (ALL), but such risk-adapted therapy has lagged in AML. This may in large part be due to the currently used treatment approaches in AML which are already extremely intensive, thus leaving little room for further intensification with currently available, conventional chemotherapy.

In this setting of being between the Scylla and Charybdis, hematopoietic stem cell transplantation (HSCT) has been considered to be an alternative therapeutic approach of both intensifying anti-leukemic agents in the form of myeloablative doses of chemotherapy and/or total body radiation, but also providing potential anti-leukemic immune responses. An important consideration for allogeneic HSCT including all types of donors is the significant short-and long-term toxicities. The balance between toxicity and survival outcome thus creates the need to carefully and prospectively determine significantly high risk categories of AML in which HSCT should be clinically tested.

At the current time, favorable prognostic groups provide for more flexibility in terms of risk-directing treatment modalities. For these patients, the issue of withholding HSCT until after relapse has become widely applied in clinical trials throughout the world. Nevertheless, with event free survival percentages still in the 60% to 70% range, this group provides little opportunity for significant dose de-escalation. An exception to this has been children with Down syndrome, particularly under age 2, with AML, who experience a survival outcome of 75% to 85% with less intensive treatment approaches. Another exception is acute promyelocytic leukemia (APL), in which molecularly targeted therapy with agents, such as all-trans-retinoic acid and arsenic, has led to significantly improved outcomes.

The identification of prognostic factors in AML may thus have less of an impact on treatment decisions and outcome than for what was originally hoped or anticipated. A key element for AML prognostication therefore lies in our ability to link those risk-based factors to the development and clinical testing of biologically targeted therapies that are cytoreductive as well as leukemic stem cell selective. Those therapies will also need to address both host and leukemic cell characteristics.

Historically, host factors such as gender, age, race and constitutional abnormalities have been associated with different outcomes for patients with AML. In most instances, such factors have not been sufficiently strong prognosticators to directly stratify pediatric patients. For example, although females appear to have slightly better outcomes than males, the effect is not so strong as to provide grounds for changes in therapeutic approaches [1, 2] . Most modern, pediatric studies have also not observed significant differences according to age, if patients are all treated similarly [1, 2] . Race, particularly being African-American, on outcome has been demonstrated to be an adverse prognostic factor in studies in which treatment was the same [3] . Another host factor that appears to affect outcome, particularly treatment-related mortality, is the patients initial body mass index (BMI). In a retrospective study, Lange et al., showed that underweight or overweight patients were less likely to survive due mostly to treatment-related infection [4] . Other host factors, such as polymorphisms in detoxification enzymes, such as GST theta, have been shown in retrospective analyses to be associated with worse outcome due to excess toxicity [5, 6] . Even if proven to be the case in a prospective analysis, there will be a need to further evaluate in clinical trials how to optimally alter initial therapy for such patients without reducing anti-leukemic efficacy. As noted above, constitutional trisomy 21 has been observed to have a significantly favorable influence on outcome for young children with AML, resulting in dosereduced treatment regimens [7] [8] [9] [10] .

Does "Response to Therapy" help to direct specific alternative approaches to treatment? The conventional measures of response to induction therapy, such as morphologic remission, have been demonstrated to predict subsequent outcome. For example, patients who have AML refractory to induction therapy, i.e., primary induction failure (>20% leukemic blasts), have a significantly worse prognosis than those achieving a complete remission [2, 11] . However, those with "intermediate" responses, i.e., between 5% to <15% leukemic blasts, have a survival similar to those patients with < 5% leukemic blasts at the end of induction therapy [2, 11] . While these data are important prognostically, they do not lead to an obvious approach to effective alternative therapies.

A more sensitive measure of response has been that of determining minimal residual disease (MRD) by either molecular or flow cytometric methods. The RT-PCR detection of t(15;17) transcripts in APL without morphologic evidence of leukemia has been shown to be predictive of eventual relapse at an early enough stage to provide time for alternative therapeutic intervention [12] [13] [14] . The detection of other molecular alterations has been less straightforward. For example, the detection of transcripts for the fusion product of the t(8;21) translocation have been observed many years after treatment in patients still in remission, suggesting that this translocation may be an early molecular alternation in leukemogenesis but insufficient for complete malignant transformation [15] . In addition, the percentage of patients with AML that have specific gene mutations or alternations that can be easily assayed is relatively low, limiting the application of this approach.

An alternative method of MRD detection that is applicable in most patients with AML is the use of flow cytometry to identify cells in the bone marrow or peripheral blood that have aberrant expression of cell surface markers characteristic of leukemic blasts. The use of flow cytometric detection of MRD is sensitive in the 1 leukemic blast per 1,000 to 10,000 normal nucleated cells. MRD detection by flow cytometry has been shown to be a significantly strong predictor of relapse in adult and pediatric AML [13, 16, 17] . Our analyses of 252 patients uniformly treated on studies from the Children's Cancer Group (CCG) showed that 16% of patients in complete remission had occult leukemia detectable by a three-color based flow cytometric method. The relapse-free survival for this group of patients positive for MRD was only 35% compared to 65% in the MRD negative group, thus demonstrating a 5-fold increased risk of relapse [3] . Importantly, the median time to relapse for patients who showed MRD positivity was 173 days; this time period would certainly provide sufficient opportunity to intervene with an alternative, therapeutic approach. More sensitive flow cytometric methods are now being used to prospectively examine the impact of MRD on outcome in pediatric AML co-operative group, clinical trials. However, while the early detection of MRD may predict relapse, it does not in itself, provide clues as to what alternative treatments would be most effective.

Distinctive cytogenetic changes are both characteristic of certain subtypes of AML but also strong predictors of outcome as determined across many different clinical trials. Favorable cytogenetics include t(8;21), inv(16) and t(15;17) chromosomal abnormalities. Excluding patients with APL and t(15;17) because of the already established alternative treatment approaches, the remainder favorable risk cytogenetic group represents about 17% of patients with AML [18] [19] [20] . However, there is strong evidence that there is heterogeneity of response and outcome within this group of patients and that patients with inv (16) abnormalities have a significantly better remission induction rate and overall survival than patients with a t(8;21) positive leukemia [21] . For example, POG has reported that although patients with either inv(16) or t(8;21) positive leukemia have better remission induction rates than patients with normal cytogenetics, post-remission outcomes varied between those with inv(16) versus t(8;21) [21] . The results from this POG study showed that patients with inv (16) had an approximately 75% versus 54% overall survival for those with normal cytogenetics. Although patients with t(8;21) AML had a relapse rate similar to those with normal cytogenetics, their ability to be salvaged with reinduction and post-remission therapy (usually including some form of HSCT) was significantly better than those with a normal karyotype. The overall improved outcome for patients with inv(16) or t(8;21) has led to reserving HSCT for patients who relapse after receiving only chemotherapy.

Patients with AML characterized by abnormalities of chromosomes 3, 5 or 7 have been shown to represent less than 20% of all patients but to also have an extremely poor prognosis, including a lower remission induction rate and decreased overall survival [2, 18, [21] [22] [23] . Although the t(6;9) translocation has been linked to a very poor prognosis, the low frequency of this chromosomal abnormality has made definitive conclusions impossible [24] [25] [26] [27] . Rearrangements involving the MLL gene have been associated with both favorable (those with t (9;11)28) as well as poor prognosis (those with t(4;11)18, 21-23). Larger and more recent studies have not demonstrated such a strong influence on outcome for patients with MLL rearrangements, making it less compelling as a means upon which to base risk-stratified, alternative treatment.

Thus, the use of cytogenetics along or in combination with response criteria represents an important approach to risk stratification for patients with AML. In addition, while the use of HSCT appears to be avoidable in CR1 for patients in the favorable risk category, the efficacy of HSCT in patients with high risk AML is not yet established. Further, the risk stratification based on cytogenetics and/or response to induction therapy, does not apply very well to nearly 60% of patients with normal karyotypes.

Activating mutations involving the gene encoding the receptor tyrosine kinase, FLT3, represent the most commonly identified molecular defects in AML. Such mutations are heterogeneous but are characterized primarily by two different classes: 1) those resulting in an internal tandem duplication (FLT3/ITD) and 2) those representing point mutations at amino acid position 835/836 within the tyrosine kinase active site [29] [30] [31] [32] . The FLT3/ITD represents approximately 12-15% of pediatric AML cases, 20-25% in young adult AML and up to 35% in older adults with AML [29, [33] [34] [35] [36] . Importantly, FLT3/ITD are most common in patients with normal cytogenetics, although there appears to be a significant percentage of patients with APL whose leukemia has the FLT3/ITD mutation [37] [38] [39] . The kinase domain point mutations occur in approximately 7% additional cases and appear to be associated with a lower white blood cell count and lower relapse rate [30, 40] . Although both classes of mutation result in resistance to chemotherapy and cytokine-independent leukemic cell survival, only the FLT3/ITD mutations have been strongly correlated with a poor outcome. It remains unclear why there is such a difference in outcome measurements between these two classes of FLT3 mutations [41] . Furthermore, because FLT3/ ITD mutations have been correlated with higher presenting white blood cell count, this historically important prognostic factor may be in part explained by this biology [2, 30, 40, 42, 43] . In addition, activating mutations have also been observed in other members of this class of receptor tyrosine kinases, including c-fms and c-KIT, but at much lower frequencies [35, [44] [45] [46] [47] [48] . Although such mutations have also been shown to produce chemotherapy resistance and cytokine-independent leukemic cell growth and survival, [49, 50] the number of patients with AML that expresses these mutations is low enough that definitive outcome measures have been difficult to obtain.

The overall survival for patients with FLT3/ITD positive AML has been shown in several recent studies to be approximately 30% compared to about 45% for those without the FLT3/ITD [33, 34, 40, 51, 52] . While this difference is significant, although not dramatic, these studies have also identified a subclass of patients with a high ratio of FLT3/ITD to wild type allele that has a particularly dismal prognosis [40, 52] . One study reported that patients with a high FLT3/ITD to Wild Type allelic ratio (i.e., >0.78) had a 0% overall survival compared to 60% for patients with an allelic ratio of <0.78.40. Similar results have been reported in pediatric studies where a high FLT3/ITD to Wild Type allelic ratio predicts a 2.7 fold increased risk of relapse in a multivariate analysis of data from the CCG-2961 trial. Of note, the presence of a high FLT3/ITD to Wild Type allelic ratio predicts a worse prognosis even in the relapse setting (Meshinchi et al., unpublished results).

The determination of an allelic ratio is influenced by the percentage of blasts in a bone marrow sample and the outcomes that have thus far been reported have not been based on an analysis of sorted leukemic blasts. Nevertheless, the results thus far have been compelling and strongly suggest the development of therapies directed toward specifically inhibiting either the FLT3/ITD receptor directly or downstream signaling events resulting from the expression of the mutant receptor.

Mutations in CEBP/alpha, a transcription factor regulating myeloid differentiation, have been associated with a favorable prognosis [53, 54] while altered expression of AF1q, a fusion partner in MLL gene rearrangements has been linked to poor outcomes [55] . Mutations in the nuclear pore complex protein, NPM, have recently been identified in patients with normal cytogenetics and associated with a favorable prognosis [56] [57] [58] [59] . Increased expression of telomerase, [60] vasoactive endothelial growth factor (VEGF)61 and the Wilms tumor (WT1) gene [62] [63] [64] [65] have all been linked to poor prognosis. However, none of these factors have achieved sufficient strength in terms of predicting outcome or targeting therapy development to have yet altered risk-based treatment stratification.

Microarray RNA expression analyses of leukemic blasts has proven to effective at subclassification of different AML subtypes. In addition, this type of analysis is proving to be a potentially important approach to further identifying risk categories as well as possibly new therapeutic targets.66 For example, Locayo et al. used RNA microarray expression analysis of samples from the POG 9421 trial to evaluate the risk of relapse in a cohort of patients with FLT3 mutations [67] . Of note, the results point to significant heterogeneity within the population of FLT3 mutant AML. For example, a high RUNX3 and low ATRX gene expression ratio showed a 0% EFS while a low ratio predicted a 70% EFS. An intermediate ratio of RUNX3 to ATRX expression showed a 37% EFS, suggesting that lower expression ratios correspond to better outcomes. Another report by Yagi et al. identified a group of 35 genes whose expression pattern was predictive of outcome [68. Studies such as these will likely increase in number over the next several years. Similarly, the use of proteomic and epigenetic profiling are also at the early stages of investigation in pediatric AML. As more of these types of studies are published, it will be important to determine whether the numbers of similar patients who are uniformly treated and analyzed in a prospective fashion are sufficient to warrant the conclusions. In addition, validation of the results in independent data set is also ultimately critical for such models that predict clinical outcomes.

While the question of whether "modern" risk-directed therapy is possible in pediatric AML may be in part semantic, the issues underlying this question are both a serious critique of current practice and a challenge for future studies. Prognostic factors have, for example, had limited use thus far in patients with AML. Some success has come out of identifying relatively small favorable and unfavorable risk groups. In the former, the use of allogeneic HSCT in CR1 is not recommended, thus saving some patients from the short-and long-term complications related to the transplant procedure. In addition, young children with DS and AML now universally receive less intensive and toxic treatment regimens without compromising outcomes. Our ability to impact on the treatment and outcome of patients with unfavorable AML has been quite limited. The recommendation to perform any type of allogeneic HSCT is a reflex strategy from the school of intensification with a potential immunologic twist. However, this approach has not been adequately tested in a controlled fashion in prospective clinical trials. Patients in the intermediate risk category have also seen little benefit from riskbased therapy.

In many respects, the goal toward risk-based therapy in AML remains essentially untapped. In fact, in most regards, this goal may not even be one of the critical questions for those investigators trying to improve outcomes for patients with AML. Instead, one should rather work toward identifying survival pathways that are distinctive for subtypes of AML with the goal of developing selective and effective therapeutic approaches. The example of APL is an important one to remind of us of this point as well as how high risk features can be therapeutically exploited and thereby eliminated. Prognostic factors, inherently linked to the treatments being tested, should be sought not for the ability to predict outcome, but for the chance to identify a more effective treatment. In the quest to find better therapeutic hammers, we must also focus our attention on the variable number and types of nails that characterize the extensive heterogeneity of AML. [1] and the median age of multicenter trial patients approaches 60 years [2] , the therapeutic outcome in the older patients remains constantly inferior to that in the younger patients. Thus, in the combined 1992 and 1999 trials by the German AMLCG enrolling 1834 patients the overall survival at 5 years is 33% in patients of 60 years. Corresponding data of relapse-free survival are 34% versus 15% [1] . There is no major difference compared to data from the CALGB published 12 years ago [3] or to recently results from the ECOG [4] . As explanations for the age related difference in outcome, (A) undertreatment in the older patients, (B) differences in the risk profiles and (C) a different role of risk factors at older age must be discussed.

(A) Undertreatment might be due to common and unavoidable age-adapted dosage and scheduling of chemotherapy. If patients are randomly assigned to induction either by TAD (standard dose thioguanine/ araC/ daunorubicin) followed by HAM (high-dose araC/ mitroxantrone) or to induction by HAM-HAM there is no difference between the two arms in overall or relapse-free survival, although the araC dosage given in the two randomized groups differs by factor 2 [3] . This result was equally seen in the younger as in the older patients whose chemotherapy dosage was age-adapted. The lack of dose-response once a certain chemotherapy dosis is achieved does not support undertreatment as an explanation. (B) In the 1834 patients with de-novo AML enrolled in the combined AMLCG 92 and 99 trials favorable karyotypes were less frequent in the older patients (7% vs. 14%) and unfavorable karyotypes more frequent (24% vs. 20%) than in younger patients (p<0.001) being the only differences in risk factors to the advantage of the younger group [5] . The striking differences in outcome again are not completely explained by these comparatively small age related differences in risk profiles. (C) For the above population of 1834 patients with de-novo AML we compared the effects of different risk factors on the remission duration in younger and older patients. As shown in figure 1 there is a general trend to a loss in discriminative power of risk factors in the older patients even significant for WBC, day 16 bone marrow blasts, and age within the age group.

We conclude that defined risk factors change their prognostic impact in older age which appears to be part of the hitherto unexplained age factor in AML. 

It is generally accepted that acute myeloid leukemia (AML) is propagated by a rare subset of cells, the leukemic stem cells (LSC) which are able to initiate and to sustain the disease. In this model the malignant clone is organized in a hierarchy, in which the progeny of the LSC forms the leukemic bulk population, which itself, however, has lost the capability to maintain leukemic growth.

Experimental evidence for such a hierarchy of the malignant clone, resembling the known hierarchy of normal hematopoiesis in many aspects, has come from functional assays, characterizing the leukemic stem cell and its progeny in functional in vitro and in vivo assays, the latter one using murine xenograft models. The observation that AML originates and is maintained by leukemic stem cells implicates that the eradication of this rare subset of cells will be necessary for the eradication of the malignant clone and ultimately for the cure of patients. The development of innovative therapies targeting the leukemic stem cell while sparing its normal counterpart, however, has been hampered by the similarities between normal and leukemic stem cells with regard to their molecular profile, but in particular also with regard to their surface characteristics. Therefore characterization of leukemic stem cells and their potential differences compared to normal stem cells are important for our understanding of the process of malignant transformation, but also for the development of novel therapeutic approaches, which aim at specifically targeting the LSC in patients with acute leukemias.

The development of appropriate functional in vitro and in vivo assays has opened the way for a detailed understanding of normal hematopoiesis and the characteristics of normal hematopoietic stem cells (HSC's). These data have shown that normal hematopoiesis is sustained by a rare subset of cells, the hematopoietic stem cell, which is characterized by the expression of certain surface antigens such as CD34 or AC133, the expression of members of the ABC transporters such as ABCG2 and its ability of self -renewal and differentiation into all the different hematopoietic cell lineages [1] . Earlier data have shown that human HSC are able to form colonies in methylcellulose after 6 weeks of stromal cell based liquid cultures (so called long-term culture initiating cells, LTC-IC)(LTC-IC assay) in vitro and to induce long-term repopulation of sublethally irradiated and immunocompromised NOD/SCID mice (SCID repopulating cells, SRCs) [2] [3] [4] [5] . The progeny of HSCs, the clonogenic progenitors, still have clonogenic potential in methylcellulose in vitro (CFCassay), but have lost repopulating activity in vivo (Fig.1 ). The adaption of these different assays to human leukemic cells has convincingly demonstrated that in acute leukemias the vast majority of leukemic cells does not show major proliferation and has even lost clonogenic potential in vitro. Only a small number of cells of the leukemic population (about 1% of the total population) is clonogenic (AML -Colony Forming Units, AML-CFU) and by far a less frequent subset of cells (1 in 106 cells) is able to initiate leukemic engraftment in the NOD/SCID mouse model (SCID leukemia initiating cells, SL-ICs) [3] . These data have strongly suggested that leukemias are organized in a hierarchy similar to the normal hematopoietic system. Furthermore, these in vivo assays have shown that the leukemic stem cell as well as its normal counterpart resides in the CD34+/CD38-/lin-compartment, expresses AC133, HLA-Dr as well as CD71, sharing these properties with normal HSC. These data underline the difficulty to find robust markers which would facilitate the discrimination between normal and leukemic stem cells.

It was shown previously, that patients with AML show a mosaïque of normal as well as leukemic stem cells in their bone marrow at the time of diagnosis and even in morphological complete remission after chemotherapy [6, 7] (Fig.2) . For the understanding of the disease but also and in particular for the development of innovative therapies a separation of the leukemic stem cells from normal HSC's would be a major step. Efforts have been made to identify surface markers that would help to distinguish normal and leukemic HSC. Blair et al. could demonstrate the efficient kill of AML clonogenic progenitors and AML stem cells in the different in vitro assays and the NOD/SCID repopulating assay in a subset of patients with AML, whereas normal HSC's were spared by the immunotoxin, suggesting differences in IL3-R mediated toxicity between normal and leukemic stem cells [10] (Fig.3) . These data demonstrated that differences between leukemic and normal stem cells can be transferred into treatment strategies and the DT(388)IL3 immunotoxin is currently investigated in phase I/II trials in patients with AML [11] . In another study Guzman et al could demonstrated that the combination of the anthracycline idarubicine with the proteasome inhibitor MG-132 efficiently killed leukemic stem cells from patients with AML as approved in vitro and in the NOD/SCID mouse model in contrast to normal cord blood derived CD34+ stem cells [12] .

Although much progress has been made in characterizing leukemic stem cells in patients with acute leukemia a robust marker allowing the separation of normal and leukemic stem cells in the daily clinical setting is still missing. First approaches to develop treatment strategies aiming at targeting the leukemic stem cell have been developed and are currently tested in Phase I/II trials, but a more detailed understanding of the biology of leukemic stem cells will be necessary for the establishment of novel therapeutic concepts in acute leukemia. Recent years have shown that the nature of the leukemic stem cell can be even more complex and that not only hematopoietic stem cells can be transformed into LSC's, but also committed progenitor cells, which re-gain 'stemness' characteristics by appropriate oncogenes and then fulfill the criteria of a LSC [13, 14] . The rapid progress in the molecular and functional characterization of leukemic stem cells, however, will ultimately pave the way to a detailed understanding of the nature of leukemic stem cells and to the design of therapies which avoid toxicity to normal hematopoietic and nonhematopoietic stem cells, but efficiently eradicate the LSC. Retrospective analysis of collaborative group data on older patients (>60 years) with AML demonstrates that survival at 5 years has changed very little in the last 20 years. In spite of recruiting in excess of 2500 patients to 2 large consecutive trials the MRC has not been able to improve outcome overall. Such patients represent on the one hand the challenge of a more chemoresistant disease as characterised by a higher proportion of adverse karyotypes, more frequent expression of resistance proteins, more disease which is secondary to previous chemotherapy or Myelodysplasia. On the other hand patients co-morbidity and other less well defined patient related factors result in higher treatment related mortality. The AML11 trial attempted to improve induction by comparing DAT vs ADE vs MidAc, but it was not possible to improve on the traditional DAT regimen. In an effort to define the optimal length of treatment 6 courses were compared to a total of 3 -again without resulting in a better outcome. Adding maintenance with Interferon for 12 months did not reduce the risk of relapse (1).

The AML14 trial was designed to compare an intensive approach versus a non-intensive approach where there was uncertainty which approach to take. Of 1600 patients who entered the trial only 10 were randomised between the options suggesting that Physicians or patients had a degree of certainty about which approach to take. Within the intensive option the main question was to evaluate the role of the Pgp modulator PSC-833. Since the use of PSC-833 required modulation of the Daunorubicin to 35mgs/m2, the opportunity was taken to compare the standard Daunorubicin dose (50mgs/m2) with 35mgs/m2). A standard dose of Ara-C (200mgs/m2/day) was compared with 400mg/m2/day. Finally, as a next stage to define the total number of treatment courses needed, so 4 courses was 600; 896; 1264; 255; respectively. With a median follow-up of 35 months no differences are observed between any of the comparisons (2). Many older patients do not wish to have, or are not considered fit for, an intensive chemotherapy approach. Population or reimbursement based data illustrate that a higher proportion of patients in the population are not given parenteral therapy aged 65-74 years is 49% to 93% in patients over 85 years (3) .

As the demographics of the older population changes, more cases of AML will occur many of who will fall into the category of unfit for intensive treatment. Apart from developing better treatments for patients suitable for an intensive treatment, there requires to be non-intensive options for the less fit and objective validated criteria which define who will benefit from an intensive approach. However there is also a question as to whether traditional trial designs involving large numbers of patients is fit for purpose when several new approaches need to be tested.

The imminent NCRI AML16 Trial aims to address a number of these issues.

Based on examination of factors associated with a more successful outcome in the AML11 trial a group of patients could be identified who seemed to benefit from an intensive approach with respect to remission and survival. A risk index based on cytogenetics, de novo of secondary disease, presenting WBC, age and performance score. These factors provided an index which could segregate who has a 55% a 40% and a 15% of being alive at 12 months and 25%, 13% and 7% at 3 years. This was prospectively validated in the intensive arm of the AML14 trial (4). New options for treatment will be evaluated in AML16. Clofarabine is a novel nucleoside analogue which has been designed to encapsulate the advantages of Cladrabine and Fludarabine which being less susceptible to deamination. In an unrandomised phase 2 trial in patients regarded as unfit for intensive chemotherapy. Sixty-two percent of 29 patients cleared marrow blasts with complete remission being seen in 52% (5) . Although well tolerated the chosen dose (30mg/m2) was myelosuppressive. Subsequent patients were treated at a 20mgs/m2 daily dose which was associated with less side effects and still retained efficacy with a remission rate of 45%. The AML16 trial will compare Daunorubicin/Cytoside vs Daunorubicin Clofarabine for the first 2 treatment courses. In addition Gemtuzumab Ozogamicin will be tested in course 1 of treatment. It is not clear how many courses of treatment in older patients is optimal. The AML14 demonstrated no benefit of a 4th course when compared with 3 courses. AML16 will address the question of whether or not 3 courses is superior to 2 courses.

Few trials have targeted older patients who are not fit for an intensive treatment approach. These patients are likely to increase as a result of demographic change and since no adequate treatment is available there is increasing interest and willingness to offer studies involving novel agents. The non-intensive arm of AML14 showed that Low Dose Ara-C is superior to best supportive care, but only for patients with intermediate risk cytogenetics. Several new agents/ combinations are available to test which provides opportunities for this patient group. However a more sophisticated trial design is required to scan for hopeful agents. The AML16 trial will use a "Pick a Winner" design by conducting several randomised phase 2 comparisons in this poor risk group to aim to improve 6 month survival compared with Low dose Ara-C.

This approach is initially designed to test novel agents or combinations in a randomised fashion with the aim of identifying opportunities that look to have a prospect of improving remission rate and survival over the 6 month term. There is a risk of falsely identifying a "winner" which is in fact a "loser" or visa versa. This has to be balanced against the traditional approach of carrying on with comparisons for too long. This pre-supposes that novel approaches have the potential to improve remission from the 18% achieved with Low Dose Ara-C to the target of 30%. Remarkable achievements in improving the cure rate for childhood acute lymphoblastic leukemia have occurred over the past four decades. Today close to 80% of children are alive without disease many years after completing therapy. Advances in cancer genetics have uncovered some of the acquired genetic mutations that are associated with leukemia cells but many steps in malignant transformation remain obscure. The ability to analyze globally the level of RNA transcripts within tumor cells (gene expression or microarray profiling) has provided an opportunity to explore cancer pathogenesis in greater detail. Gene expression profiles have been identified that define blast phenotype, genotype and clinical behavior. The study of blasts at relapse compared to initial diagnosis has identified pathways that might serve as novel therapeutic targets and has provided insights into the cellular origin of clones observed at relapse. Advances in the treatment of childhood acute lymphoblastic leukemia (ALL) serve as a paradigm to improve outcome for all human cancers. However new challenges now face clinicians. Some children are being over-treated so that others can benefit from intensified therapy. In addition, 20% of patients suffer a relapse and their outcome is dismal. To overcome such obstacles biological insights into the pathogenesis of childhood ALL are needed. Acute leukemias, like other cancers, are a group of somatically-acquired, clonal genetic disorders that emanate from a single cell. In the case of childhood ALL these genetic lesions are well characterized, such as gains and/or losses of whole chromosomes, as well as translocations. These cytogenetic findings provide significant prognostic information and are now used routinely to stratify patients into treatment protocols that differ in intensity, thus balancing efficacy and toxicity. For example, patients with adverse cytogenetic findings such as extreme hypodiploidy (DNA index <0.81 or <44 chromosomes), MLL translocations or the t(9:22) generally receive more intensive treatment than those with favorable cytogenetics such as hyperdiploidy with trisomies 4, 10 and 17 and the TEL-AML1 fusion..

In spite of these associations, the underlying biology that drives malignant transformation and drug sensitivity remains unknown. Although these sentinel changes may recapitulate some components of the malignant phenotype in experimental systems it is clear that multiple steps, may be needed to transform a pre-malignant stem cell into a frank leukemia. Current research is now focused on defining these additional transforming events as well as the downstream consequences of known genetic rearrangements.

The field of cancer genomics has undergone a remarkable revolution that was aided by technical breakthroughs that led to the sequencing of the human genome and the ability to analyze comprehensively the expression of all genes simultaneously using DNA "chips" or microarrays [8, 9, 13] . More recently the introduction of high density "SNP chips" will no doubt greatly refine single nucleotide polymorphism (SNP) mapping and analysis of gains and losses at chromosomal sites [12] .

The development of microarray technology was met with great enthusiasm by cancer investigators six years ago, but like other breakthroughs, initial enthusiasm has been tempered by the practical realities of experimental design, reproducibility, inadequate bioinformatic tools and pitfalls with the unavoidable "false positives" associated with analyzing expression using over 30,000 gene probes.

Early proof of principle experiments showed that microarray patterns could reliably distinguish morphological and immunophenotypic subtypes of ALL [7, 14] . Importantly investigators first piloted the tools needed to analyze the vast amount of data associated with such experiments. Subsequent experiments examining a large number of cases (327 samples) showed that an unbiased analysis of gene expression data alone led to the definition of seven major subgroups of ALL. These subgroups correlated with immunophenotype (T-ALL), translocations status (BCR-ABL, E2A-PBX1, TEL-AML1 and MLL rearrangement), hyperdiploidy as well as a seventh group without a definitive molecular or cytogenetic characteristic [14] . Thus these experiments clearly defined the "downstream consequences" of major cytogenetic changes and provided clues to the marked differences in outcome among these subgroups.

Although numerous clinical and biological features are used to predict outcome and stratify patients, most children who fail therapy were initially classified in a favorable risk subgroup. These findings have led many investigators to study the ability of gene expression profiles at diagnosis to predict outcome. In the large data set mentioned above expression profiles predictive of relapse could only be defined for T-ALL and hyperdiploid cases [14] . Similarly Mosquera-Caro et al. reported novel gene sets that correlated with outcome using samples from children treated on legacy Pediatric Oncology Group protocols [11] . However there was little to no overlap between the gene sets from the two studies possibly due to differences in treatment between the two cohorts. The inability to identify common predictive gene sets among these and other studies is in sharp contrast to the stable predictive character of variables such as age, white blood cell (WBC) count, cytogenetics and response to therapy. These latter features have consistently shown prognostic significance regardless of differences in treatment across studies, so should investigators expect less from gene expression studies?

To address the issue of treatment differences our laboratory has performed oligonucleotide microarrays on 99 B-precursor patients treated on Children's Cancer Group CCG 1961 protocol for high risk ALL to identify gene expression signatures that correlate with early response to therapy as judged by morphological assessment of blast content in day 7 and day 14 bone marrow samples, and event free survival [10] . We were unable to identify a gene signature that reliably correlated with the day 7 bone marrow response suggesting that many biological pathways are associated with early blast regression. This finding is in agreement with recent results from St. Jude Children's Research Hospital where investigators were also unable to define a signature that correlated with end induction minimal residual disease (MRD) [5] . However Cario et. al did discover a gene expression profile that correlated with detectable MRD at a much later time point, week 12, and likewise we could also identify a highly significant set of genes associated with an M2 or M3 bone marrow at day 14 [3, 10] . Thus a small subgroup of patients with a very slow response might be identified by expression profiling on the diagnostic sample. Importantly, expression of 47 probe sets were significantly different in patients who were in complete remission for more than 4 years compared to cases that experienced a relapse within the first 3 years (FDR <0.05%). Logistic regression analysis showed that each of the 47 probe sets had additional prognostic value when accounting for age and WBC count. At least one third of these genes were significantly predictive of outcome in the independent expression data set of Mosquera-Caro that used a smaller probe set with U95Av2 microarrays [11] .

The cellular origin of relapse should be considered when attempting to use gene expression profiling to predict relapse. If relapse is due to the emergence of a rare subpopulation of cells with a completely different expression profile compared to the bulk of tumor cells at initial diagnosis then it is unlikely that expression profiling of diagnostic samples will reliably predict relapse. To address this issue and to uncover mechanisms leading to treatment failure we have performed microarray analyses on leukemic cells from a cohort of 60 patients who experienced their first bone marrow relapse and were treated on the Children's Oncology Group AALL01P2 trial [1] . We also extended these studies to include a matched pair analysis of marrow samples from both initial diagnosis and initial marrow relapse (35 pairs) [2] . Given the prognostic importance of timing of relapse [4, 6] , we identified genes which accurately distinguished early (<36 months from diagnosis) from late ( 36 months) marrow relapse. We found a large number of genes that were differentially expressed between early and late relapse [1] . Functional classification demonstrated that genes playing a role in cell proliferation, DNA repair and cell survival were prominently up-regulated in the early relapse group. A subset of genes was detected that correlated with response to therapy as evidenced by induction failure and MRD after the first block of treatment.

Unsupervised analysis of the relapse pairs showed that early relapse samples were more likely to align themselves next to their respective initial diagnosis partner but higher correlation coefficients were observed between late relapse/initial diagnosis samples [2] . One hundred and twenty one probe sets (45 high at diagnosis, 76 high at relapse) were identified in a pair-wise analysis to be significantly different at relapse compared to diagnosis (FDR <10%). Many genes involved in cell cycle regulation (e.g. cyclinB1), protein biosynthesis, DNA replication and repair (e.g. topisomerase IIA) and anti-apoptosis (e.g. survivin) were differentially regulated at relapse. A similar analysis was performed after excluding the three patients with T-cell ALL. Using a similar cutoff of FDR <10%, 78% of the genes were common. Since there are significant differences in outcome between early vs. late relapse, these relapse pairs were examined separately. Seventy three probe sets (FDR<10%) were differentially expressed between the early relapse pairs (N=23) but there were no gene expression sets that were significantly different in the late relapse patients compared to their initial diagnosis sample (N=12).

Our inability to define an expression signature that distinguishes diagnosis and late relapse and the wider correlation co-efficient of such pairs suggests that the cell of origin may differ between early vs. late relapse. Drug resistant clones that represent a very small minority of cells have been identified at initial diagnosis using molecular techniques designed to detect specific mutations in a large number of background cells. In this case the relapsed clone may share more characteristics with the large number of drug sensitive cells but differs in a small number of critical pathways. This model fits with our observations for early relapse. In contrast late relapse may be due to secondary changes in a preleukemic stem cell as has been documented in some cases of relapsed TEL-AML1+ ALL [15] . These secondary events may result in broader changes in the transcriptome such that diagnosis and relapse may be dissimilar.

In summary a molecular taxonomy of leukemia is being developed through the use of microarray technology. Experience to date has shown that gene expression profiling can be used to classify subtypes of ALL but is unlikely to replace conventional methods such as fluorescence in situ hybridization and flow cytometry to classify ALL in the near future. Predictive signatures that correlate with outcome have been reported by different investigators but little overlap in gene sets have been observed to date. Without further validation these signatures cannot be incorporated into risk assignment. However this technology has led to the identification of candidate pathways that may be directly involved in mediating drug resistance. However, optimizing the chemotherapeutic regimen by introducing less toxic but equally effective elements together with better supportive care strategies will reduce treatment related mortality and thereby improve survival in small steps.

At present, 55%-60% of children with AML are long-term survivors; whereas in the 80ies, the percentage was below 50% [5, 11, 12] . This improvement was mainly achieved by an intensified chemotherapy and an essentially advanced supportive care. In study AML-BFM 93, outcome in high-risk (HR) patients was improved by intensification with HAM (high-dose cytarabine and mitoxantrone) [6] . As toxicity of the HAM block proved to be tolerable, we introduced this therapy element in study 98 for all patients (excluding M3) in order to improve as well the prognosis of standard risk (SR) patients. In addition, we compared the impact of the six-week consolidation treatment with seven different drugs as traditionally given in the BFM studies with two short therapy cycles with higher dose intensity. Furthermore we tried to reduce initial toxicity through support of G-CSF under controlled conditions. Patients: Between 7/1998 and 6/2003, 473 children and adolescents Treatment: After induction with AIE (cytarabine, idarubicin and etoposide) all patients (SR and HR, excluding FAB M3) were treated with HAM (high-dose cytarabine 3g/m/12hx3 days and mitoxantrone 10mg/m/dayx2 days), which, in the previous study 93, was given for HR patients only. Subsequently, all patients were randomly assigned to receive either the 6-week consolidation as in the previous studies [6] or two short therapy cycles including higher doses of cytarabine but the same cumulative dose of anthracyclines. The following therapy elements, intensification with high-dose cytarabine/etoposide and maintenance, were similar in studies 93 and 98 (for details see legend of Figure 1 ). Allogeneic stem cell transplantation from a matched family donor was restricted to HR patients only.

All patients (except those with >5% blasts in the bone marrow on day 15 and those with M3) were eligible for randomization to receive or not to receive G-CSF (5g/kg/day) on days 15-20 after AIE induction and 2nd induction with HAM. Overall results of study AML-BFM 98 are shown in Table 1 :

General results in study 98 are in the same range as in study 93 [5] and are comparable to results achieved in other study groups with intensive therapy regimen in children [11, 12] . Remission rate (CR) and 5-year survival improved in study AML BFM 98 compared to study AML-BFM 93 (CR: 88% vs. 82%, p .01; p-survival: 62%, + 3% vs. 58%, + 2%, p logrank .03, Figure 2 ), however, 5-year pEFS and disease free survival (DFS) were in the same range as in study 93 (pEFS 49% + 3% vs. 50%, + 2%, p logrank .77, pDFS 57% + 3% vs. 61%, + 3%, p logrank .32) [5] .

Compared to the previous AML-BFM trial the early death rate could be further reduced in AML-BFM 98 (7.4% in trial AML-BFM 93 vs. 3.2% in AML-BFM 98, p-(Fisher) .004). This was mainly due to improved supportive care and treatment experience [7] .

Results according to different risk parameters (gender, age, leukocyte count, FAB type, CNS involvement and karyotypes) did not show significant differences in pEFS in studies AML-BFM 93 and 98.

In study 93, the intensification with HAM [6] was the main reason for the significantly improved prognosis in HR patients. Therefore, the same intensification with HAM was given to SR patients in study AML-BFM 98 [6] , however, without improvement for these patients compared to study 93 (SR patients [FAB M3 excluded] in study 98 compared to those of the previous study AML-BFM 93: CR rate 93% vs. 89%, p(chi) .24, 5-year pEFS 62%+4% vs. 67%+ 4%; plogrank .37). Eight (4%) SR patients died in CCR (study 93: 6 patients = 3%). Results in HR patients were also similar to those of the previous study.

Our data indicate that it was possible to improve prognosis in SR patients in the 80ies by intensification of initial chemotherapy [8] . In the 90ies, prognosis could be improved in HR patients, too, by more intensive therapy, e.g. by highdose cytarabine courses in study AML-BFM 93 -which suggests that this patient group might even benefit from further treatment intensification [6] .

Consequently, in study AML-BFM 98, also SR patients were treated with HAM. However, pEFS and p-survival in SR patients turned out to be similar to those of the previous studies 93, 87 and 83. These results had not been expected, however, they show that it is extremely difficult to improve prognosis in these patients. Analogous observations have been reported from the MRC trial. In the "MRC -good-risk group" defined by favourable cytogenetics, there was no survival advantage by allogeneic SCT in 1st CR compared to chemotherapy only [3, 12] .

New therapeutic approaches like targeted therapy or subgroup directed therapy might be able to increase survival in SR patients One example is the application of FLT3 inhibitors in FLT3-positive patients or of tyrosine kinase inhibitors for children with RAS mutations or KIT mutations [9] .

As the FLT3 internal tandem duplication (ITD) is an independent unfavourable prognostic factor in childhood AML (also in the BFM studies) [13] ; these patients, who often present with normal karyotypes, will be reclassified to the HR group. In the ongoing study AML-BFM 2004, SR patients (excluding now FLT3-ITD positive patients) are spared the treatment with HAM, aiming at improving outcome in this group by a better "good-risk" definition and by avoiding therapy related mortality associated with HAM.

Besides intensity, type and order of therapy elements are further parameters which have an impact on therapy success. The AML-BFM consolidation therapy over 6 weeks has the disadvantage of being difficult to complete in a timely fashion [4] . In pediatric and adult AML studies, therapy with at least 4 intensive, mainly high-dose cytarabine-based therapy cycles led to good results in patients with AML [1, 11] . A comparison between the results of the randomised therapy arm of two short-cycles and the 6-week consolidation arm showed similar outcome (p-survival, pEFS, [ Figure 3 ] and pDFS) by intent-to-treat-analyses and when analysed as treated. However, there were less toxic deaths in the 2-cycle arm (5 vs. 9 patients; mainly due to severe bacterial infections in aplasia). In addition, the total time for the intensive treatment phase was in median 15 days shorter within the 2-cycle arm.

More experience is needed for the 6-week consolidation which is much more difficult to handle than the 2-cycle therapy, for which strict rules can be given concerning the duration of the therapy interval between the cycles and conditions for starting the next cycle. These considerations have led to the conclusion to establish the 2-cycle consolidation in future AML-BFM studies. Results by randomization of G-CSF As adult AML studies showed that the length of neutropenia was shorter and that fewer antibiotics were needed when G-CSF was given [2, 10] , we examined randomly in study 98 whether the administration of G-CSF after the 1st and 2nd therapy block could reduce the duration of neutro-and trombopenia and the incidence of serious infections (WHO grade 3/4). Results showed that G-CSF shortened significantly the duration of neutropenia in children undergoing induction therapy for AML, but that there was neither an influence on the incidence of infectious complications nor on the 5-year-EFS rate.

One hundred thirty-one of the eligible patients were randomized to receive treatment with G-CSF and 136 patients to receive treatment without G-CSF. The duration of neutropenia (<500/ l) was shorter in the G-CSF group (after AIE induction: median 18.0 vs 23.0 days, p=.02 and after HAM: 11.5 vs 17.0 days, p=.0001). The time of thrombocytopenia (<20,000/l) was similar in both groups. No impact of G-CSF was found on the incidence of grade 2 infections (fever without an identified pathogen) and grade 3/4 infections (fever due to a microbiologically documented infection). Five-year pEFS did not significantly differ between the groups with and without G-CSF (56%+5% vs 50%+5%, plogrank .33). Further, no significant difference was found when comparing the results of children randomized for G-CSF (intent-to treat) and of children who actually received the hematopoietic growth factor. As there was no evidence of beneficial effects in our study concerning serious infections, the administration of G-CSF will not be recommended for routine use in the future.

The estimated survival is now in the range of 65%-70% for those patients who achieve remission. Results of study 98 show that survival in SR patients cannot be improved by intensification of chemotherapy with HAM. Prophylactic administration of G-CSF was of no clinical benefit. Experience of the treating physicians/hospital seems to be an important factor for prognosis [7] . As improvement of prognosis cannot be achieved by intensification of treatment alone, optimizing the chemotherapeutic regimen and supportive care strategies will reduce treatment related mortality and thereby improve survival in small steps. 

Within the past years, reduced or modified doses of chemo -or radiotherapy have been widely studied for conditioning before allogeneic hematopoietic stem cell transplantation in patients with myeloid leukemia not eligible for conventional transplantation. The main goal was to reduce the substantial treatment-related mortality in this patient population while preserving the potential curative graft versus leukemia effect. This review summarizes results of published trials using reduced-intensity conditioning (RIC) in patients with AML.

In most of the published trials conditioning contained fludarabine (90-180 mg/m2) in combination with busulfan (4 -10 mg/kg), melphalan (90-140 mg/m2) or 2-5 Gy total body irradiation (TBI). Peripheral blood hematopoietic stem cells from related or unrelated donors were used as graft source in most of the studies. Post-transplantation immunosuppression consisted of cyclosporine combined with methotrexate or mycophenolate mofetil. Although the majority of the patients were above the age of 50 years, early treatment related mortality was rather low. Nevertheless, the rate of clinical significant GvHD seemed to be comparable to conventional transplants in most of the protocols. The outcome differed between trials, but diagnosis and disease status pre-transplant significantly influenced outcome.

In summary, this approach is feasible and provides access to the curative potential of allogeneic stem cell transplantation for patients with higher age or comorbidities. Since the majority of the reports included heterogeneous patient populations, mostly with a short follow-up, randomized studies are needed to define the role of RIC before allogeneic hematopoietic cell transplantation.

Allogeneic hemopoietic stem cell transplantation (HSCT) using myeloablative chemo -or radiotherapy is able to achieve long-term disease control in patients with myeloid hematological diseases. Nevertheless, older patients and those with comorbidities experience a high treatment-related mortality, which makes them ineligible for conventional conditioning therapy. Therefore, new protocols using reduced-intensity conditioning (RIC) have been developed in order to reduce the extramedullary toxicity. The rationale for this approach is the known graft versus leukemia (GvL) -effect which is responsible for the low relapse rate after allogeneic transplant compared to conventional chemotherapy or autologous transplantation (1). This concept was convincingly supported by the effectiveness of donor lymphocyte infusions after allogeneic transplant in patients with relapsed AML and CML (2) . This has disembogued in a strategy of reducing early toxicity while exploiting the GvL-effect post transplant.

Within the past years many investigations using reduced doses of conditioning prior to allogeneic HCSCT have been published. The aim of this review is to summarize results with RIC in patients with acute myeloid leukemia. Only studies with a median follow-up of >12 months are discussed in detail.

Despite important advances in the therapy of AML the majority of patients die from their disease (3). The prognosis of older patients with AML is poor with a mean complete remission (CR) rate of 63% and the probability of remaining in CR after 4-5 years of 22% (4). Due to an increase in comorbidities like infections and impaired organ function the arbitrary age limit for intensive conditioning therapy prior to allogeneic transplantation in patients with AML is between 50 and 55 years. As the median age in AML is more than 60 years, the adequate management of AML in older patients remains a major challenge. Reduced intensity or risk adapted conditioning therapy for allogeneic HSCT might be one way to reduce the substantial TRM of older patients and thus providing the curative potential of allogeneic cell therapy.

The German Cooperative Transplant Study Group (5) published data in 113 elderly patients with AML receiving RIC prior to allogeneic HSCT. Results from a survey of different centers were summarized, which explains the heterogeneity of the conditioning regimes and post-transplant immunosuppression. Most of the patients received PBSCs from unrelated donors. Nevertheless, there was a low rate of chronic extensive GvHD and TRM was significantly dependent on disease stage pre-transplant. In this cohort of patients mostly with bad-risk features due to disease stage, age, comorbidities and prior therapy overall survival was 29 % after a short median follow-up of 12 months.

A combination of melphalan and fludarabine was tested prospectively in 34 patients with high-risk AML and MDS (6). The majority of the patients was in relapse or had progressive disease. Hemopoietic cells from related and unrelated donors were transplanted. More than one third of the patients died within the first 100 days after transplantation and the overall survival was reported to be 39% at 1 year. Those data show that the probability of achieving disease control in patients with advanced disease stages remains low.

The conditioning protocol with the lowest intensity published so far was pioneered in Seattle by R. Storb and colleagues at the Fred Hutchinson Cancer Research Center. It is a direct translation from canine studies where 2 Gy TBI with CsA and mycophenolate mofetil (MMF) post transplant led to a sustained engraftment of DLA-identical donor cells in the majority of the animals. In the study by Sweeney et al. (7) ten older patients (median age 60 years) with AML were transplanted from HLA-identical siblings according to the protocol above. Only one patient rejected the graft but recovered after mild cytopenia. The TRM was low with 11 % on day 100. After a median follow-up of more than 1 year 50 % of the patients are alive and free of disease. Feinstein et. al. showed that the addition of fludarabine can help to reduce the rejection rate in AML patients given 2 Gy TBI only (8). 18 patients (median age 59 years; range 36-73 years) with de novo (n = 13) or secondary (n = 5) AML in first complete remission were transplanted from HLA identical siblings. In two out of 10 patients who received only TBI 2 Gy a graft rejection was observed. Overall, 10 patients died (7 with relapse). Seven patients are alive without relapse. The propability of survival at 1-year is 54% (95% CI 31-78%) and the relapse-free-survival 42% (95% CI 19-66%).

In a current update, the Seattle experience using this approach in 300 patients suffering from various diseases is summarized (9). The risk of relapse is decreased in patients with chronic graft-versus-host disease resulting in a higher survival rate. In contrast, patients with acute graft-versus-host disease had a high incidence of treatment related mortality. Further strategies should therefore focus on the reduction of the incidence of acute GvHD without decreasing the graft-versus-leukemia effect.

Two further studies showed the feasibility of regimens containing drugs effective in AML (10) and the role of Campath-1H (11) again in GvHD prevention.

The benefit of allogeneic transplantation was also shown in a study from the Freiburg group (12) . 19 patients with AML not all in remission in the age group above 60 years (median 64 years) received a condition regimen containing fludarabine, melphalan and carmustin. Peripheral blood stem cells were taken from unrelated (n=12) or related (N=7) donors. 13 out of 19 patients are alive with a median follow-up of 825 days (range 595-1028 days).

The feasibility of reduced intensity conditioning protocols even in aplasia early after induction chemotherapy was shown by the Dresden group (13) . This approach is now evaluated in a randomized study.

In conclusion, RIC followed by allogeneic HSCT in AML patients results in engraftment and can achieve disease-control even in poor-risk disease. Early TRM seems low in most of the studies, but the rate of GvHD and infectious complications is comparable to that after standard conditioning. However, the conditioning regimens and patient cohorts published so far are quite heterogeneous. The next generation of clinical studies will have to randomly compare different preparative regimens in homogeneous patient cohorts to further delineate the role of RIC. (a) The German Cooperative Transplant Study Group initiated two prospective randomized protocols. The standard conditioning therapy with 12 Gy TBI and 120 mg/kg cyclophosphamide will be compared in patients with AML in first complete remission with RIC consisting of fludarabine and 8 Gy TBI. (b) The place of immediate early transplantation will be compared with conventional transplantation after remission induction chemotherapy in high risk patients. 

Our interest in ATRA + ATO reflected the possibility that the combination might allow elimination, or reduction in the amount, of chemotherapy necessary to cure newly-diagnosed APL. Here we report results using this combination for both induction and post-remission therapy with chemotherapy [gemtuzumab ozogamycin, hereafter called mylotarg(1,2)] added only: (a) if toxicity forced discontinuation of ATRA or ATO, (b) for persistence of polymerase chain reaction (PCR) positivity for PML-RAR, or (c) for reversion of a negative PCR ("molecular relapse"). Additionally, the protocol called for high-risk patients (presenting WBC>10,000/l) to receive 1 dose of mylotarg on day 1 of induction therapy. This decision reflected the low CR rate we had seen in high-risk patients given liposomal ATRA monotherapy (3).

The 39 patients we treated had a median age of 44 years and median presenting white count 6,600/l. Using the system of Sanz et al. (4), 18 patients were high risk (presenting WBC>10,000/l), 6 were low risk (WBC<10,000 and platelet count>40,000 / l) and 15 were intermediate risk (WBC < 10,000 and platelet count<40,000/l). Hereafter, we will combine the low and intermediate groups into a single "low-risk" group. Low risk patients received ATRA 45 mg/m2 in 2 divided doses daily and, beginning 10 days later, ATO 0.15 mg/kg IV over 1 hour daily. Marrow aspirates were obtained approximately weekly beginning 25-28 days after start of treatment. Once the marrow showed <5% blasts and no abnormal promyelocytes, ATRA and ATO were discontinued until occurrence of CR. Once in CR, patients received ATO at 0.15 mg/kg IV daily on Monday through Friday of weeks 1-4, 9-12, 17-20, and 25-28. They took ATRA 45 mg/m2 daily during weeks 1-2, 5-6, 9-10, 13-14, 17-18, 21-22, 25-26. Therapy concluded 28 weeks after CR date.. If either ATRA or ATO had to be discontinued because of toxicity, patients received mylotarg 9 mg/m2 once monthly until 28 weeks had elapsed from CR date.

Qualitative PCR testing using bone marrow specimens was done at CR and every 3 months thereafter for 2 years. The test could detect PML-RAR fusion transcripts present at concentrations 10(-4). If, beginning 3 months from CR date, the PCR result was "positive", a repeat test was done 2-4 weeks later. If this test was also positive, patients received mylotarg 9 mg/m2 once monthly for 3 months. If the PCR became negative, 3 more months of mylotarg were prescribed; if not, idarubicin 12 mg/m2 daily X 3 was substituted for mylotarg. Mylotarg was also to be added for clinical relapse (e.g. in an extramedullary site) occurring in the context of a negative marrow PCR. High risk patients were treated identically to low risk patients except during induction 17 of the 18 highrisk patients received chemotherapy: mylotarg 9 mg/m2 on day 1(14 patients), idarubicin 12 mg/m2 days 1-4 (1 patient), mylotarg (9 mg/m2 day 1) and idarubicin (12 mg/m2 days 1-3) (2 patients).

The CR rate was 34/39 (87%): 19/21(90%) in low risk patients and 15/18 (83%) in high-risk patients. 3 of the 5 failures died within 3 days of starting treatment, the 4th died on day 17, but presented with a cerebral infarct., and the 5th ,who presented with APL and intra-medullary breast cancer, had persistent pancytopenia due to breast cancer, which unlike the APL remained in the marrow after treatment. Time to CR was longer in high-risk patients (p=0.03) although the difference in median time to CR was only 4 days (32 vs. 28). The APLDS was noted in 6 patients (3 low risk, 3 high risk) and was judged possible in a 7th (low risk). Five patients had toxicity (1 peripheral neuropathy, 4 cardiac) that led to discontinuation of ATO and its replacement by mylotarg , and in a 6th patient cardiac toxicity prompted a 25% reduction in ATO dose; the cardiac complications are described in reference 5.

Relapse has been observed in 2 patients, both high-risk. In the first patient, molecular relapse, i.e. 2 positive PCRs one month apart, occurred 9 months from CR date and was simultaneous with clinical relapse. The second patient had a molecular relapse 1 year from CR date, and despite addition of mylotarg had a relapse in the cerebrospinal fluid 4 months later. The 32 patients remaining alive in 1st CR have been followed for a median of 13 months from CR date. Five patients have been followed for more than 2 years . Each patient is PCR negative at last follow-up. Thus, none of the 21 low-risk patients have received chemotherapy, and only 2/15 high-risk patients have received chemotherapy after CR. The durations of hematologic and molecular remission are identical in 21 of the 32 patients, and in the 11 others the median difference between hematologic and molecular remission is 2 months.

Thirty-two of the 34 patients achieving CR had PCR testing at, or about the time of, CR. Thirty-one of the 32 were positive. However 3 months later 31/31 patients were PCR negative. Subsequent rates of PCR negativity were 26/26 at 6 months from CR date, 22/23 at 8-10 months, 19/21 at 11-13 months, 12/14 at>13 months, and 4/6 at >2 years.

The results suggest that ATRA + ATO is effective treatment for newly-diagnosed APL and may obviate, or at least greatly reduce the need for chemotherapy. We are certainly aware that our median follow-up of 13 months in the 32 of our patients remaining alive in first CR can be criticized as "short". Indeed, the risk of relapse or death in CR among our historical patients given idarubicin + ATRA was 15% in the first 13 months from CR date and 27% subsequently. The issue of what constitutes adequate follow-up is a general one. For example, a paper claiming an advantage for imatinib mesylate in CML recently appeared (6), although, given the chronic nature of CML, the median follow-up of 28 months seems relatively less than the 13 month median follow-up reported here.

Burnett et al. have demonstrated that similar proportions of patients who remain in CR and patients whose disease recurs are PCR positive at CR, and that the predictive value of a positive PCR is greatest after 3 cycles of chemotherapy have been delivered (7) . Thus, there is no necessary clinical significance to the virtually 100% rate of PCR positivity in our patients at CR, given that with continued treatment all patients became PCR negative within 3 months of CR date. Nonetheless, the high rate of PCR positivity at CR is in contrast to rates seen not only with ATRA + chemotherapy (7), but in Shen et al.'s trial of ATO + ATRA used without chemotherapy for remission induction but with chemotherapy thereafter (8) . In this latter study ATO and ATRA began simultaneously, and time to CR was the same as in our study in which ATO began 10 days after ATRA. The lower rate of PCR positivity at CR in Shen at al's trial (8) suggests that it is preferable to give ATRA and ATO simultaneously.

Assuming that further follow-up confirms the activity of ATRA + ATO, several potential uses for this combination come to mind. Thus, although treatment of newly-diagnosed APL is quite successful, problems remain in treatment of older patients and high-risk patients. Using idarubicin, mitoxantrone, and ATRA, Sanz et al. noted that 6/25 of their patients age 70 and above died in remission (9), while Fenaux et al reported that 15% of patients age 60 years and above died due to complications of myelosuppression during consolidation with daunorubicin + ara-C (10). These observations have led Fenaux et al. (10) and LoCoco et al. (11) to propose attempts to reduce the intensity of chemotherapy, at least in patients with low-risk untreated APL; our data suggest ATRA and ATO may be useful in this regard. The combination, together with mylotarg as administered, here may also be useful in high-risk patients in whom success rates remain considerably below those seen in low-risk patients and who remain the focus of attempts to improve outcome (12) . Indeed a trial of ATRA + ATO + mylotarg will soon begin in the North America Intergroup.

The detailed analysis of chromosomal changes in acute myeloid leukemia (AML) has been one of the key steps in understanding the pathobiology of this disease. The translocation t(8;21)(q22;q22), first described by Janet Rowley [1] forms the fusion gene AML1-ETO, which occurs in up to 12% in all AML cases and up to 40% in the AML FAB M2 subtype [2, 3] . Patients with this subtype of AML are associated with a more favorable prognosis, but more recent data have shown that patients with this fusion gene harboring additional activating of receptor tyrosine kinases (RTK) such as c-KIT or FLT3 have a very poor prognosis [4, 5] . In this situation one key question is to which extent collaborating genetic events determine the leukemogenic process in AML1-ETO associated leukemias. More recent data in experimental models have pointed to a key role of these RTK mutations for the development of AML1-ETO positive AML. This would have clinical implications, as efficient RTK inhibitors are available and are currently tested in several phase I/II trials in patients with AML.

The challenge for our understanding of AML1-ETO induced leukemias is that several animal models clearly demonstrated that the fusion gene itself is unable to cause leukemia in vivo. This was shown in murine models using retroviral gene transfer as well as in mouse models using transgenic or conditional knockin strategies: in a conditional AML1-ETO murine model, only mice treated additionally with ENU developed AML as well as T-cell lymphoblastic lymphoma, whereas non-treated AML1-ETO mice showed only minimal hematopoietic abnormalities [6] . Similar observations were reported from a hMRP8-AML1-ETO transgenic mouse model, which developed AML as well as T-ALL/lymphoma only after ENU mutagenesis [7] , and from a murine bone marrow transplantation model inducing constitutive expression of AML1-ETO in hematopoietic progenitor cells by retroviral gene transfer [8] . In a recent report, mice targeted to express AML1-ETO in the hematopoietic stem cell compartment, developed a non-lethal long-latency myeloproliferative syndrome, but failed to develop acute leukemia [9] .

In particular, data from the ENU treated mice strongly indicated the importance of additional genetic hits in patients with AML1-ETO positive leukemia. The first experimental hint, that constitutively activated RTK's can collaborate with AML1-ETO came from a murine leukemia model, which demonstrated that coexpression of ETV6-PDGFR, causing already by itself a lethal myeloproliferative syndrome in transplanted mice, with AML1-ETO resulted in AML [10] . In addition, the model of genetic collaboration is now strongly supported by data based on the molecular characterization of AML1-ETO or other 'core binding factor' (CBF) leukemias in patients. It was shown that genetic lesions affecting transcriptional regulators such as AML1-ETO, HOX fusion genes or PML-RAR frequently occur together with activating mutations of RTKs. In contrast, genetic lesions of the same category (e.g. AML1-ETO with Hox fusion genes or FLT3-LM with c-KIT mutations) rarely occur together [11] . These observations have favoured a model, in which collaboration of two groups of genetic alterations, one affecting transcriptional regulation and hematopoietic differentiation, the other altering signal transduction cascades associated with cell proliferation, are necessary for the initiation of leukemia [12] . We recently reported on a series of 135 adult patients with AML1-ETO positive AML, analyzed for the additional occurrence of activating mutations involving signal transduction pathways (FLT3-LM, FLT3D835, KITD816, NRAS codon 12/13/61) or concurrent MLL partial tandem duplicaton (PTD). Of note, almost one third of all AML1-ETO positive patients had mutations affecting RTK's or NRAS, whereas none showed the MLL-PTD [13] . These findings were confirmed by other reports describing KIT and RAS mutations in 70% of pediatric CBF leukemias in a series of 150 patients [14] or c-KIT mutations in 48% of patients [15] . Recently it was shown that additional activating mutations of RTK's such as c-KIT clearly change the clinical outcome in patients with AML1-ETO positive AML with a significantly shortened overall and event free survival: patients with AML1-ETO and additional c-KIT D816 mutations had an overall survival of 304 versus 1836 days (p=0.006) and an event free survival of 244 versus 744 days (p=0.0027) [16] (Fig. 1 ). The first functional evidence, that AML1-ETO collaborates with an activated RTK recurrently found in patients with AML1-ETO positive leukemia, was recently provided in a murine bone marrow (BM) transplantation model [13] : when mice were transplanted with BM cells retrovirally expressing AML1-ETO or FLT3-LM, none of the animals developed a disease. However, all the animals, which engrafted with bone marrow cells co-expressing both cDNA's developed transplantable acute leukemia (Fig. 2) . Interestingly, 4 of the 7 AML cases revealed co-expression of the lymphoid marker CD4. Of note, patients with AML1-ETO positive leukemia also show co-expression of lymphoid antigens in the majority of cases [13] .

The concept of collaboration of two classes of mutations in AML might have clinical implications: in theory a therapy targeting both collaborating genetic alterations should be more effective than a therapy which just aims at one of the two leukemogenic genetic events. However, this concept is still hampered by the yet limited number of targeted therapies available. But in the case of the AML1-ETO positive leukemia collaboration of the fusion gene with activating RTK's would provide a rationale to combine chemotherapy with novel drugs targeting RTKs and downstream effectors, which have been developed and are currently tested in several clinical trials. At the moment at least four FLT3 inhibitors are in clinical trials and others are at various stages of development [17] . In the murine AML1-ETO/FLT3-LM leukemia model, AML1-ETO was not able to collaborate with a kinase dead FLT3-LM mutant and treatment with the FLT3 -inhibitor PKC412 reduced the oncogenic collaboration of AML1-ETO and FLT3-LM by 60%, pointing to a potential clinical activity of these drugs in Figure 1 : Overall survival of patients with AML1-ETO with or without c-KIT D816 mutation [16] the subset of AML1-ETO positive leukemias harboring additional RTK mutations [13] . Future trials have to prove that RTK inhibitors as part of multimodal therapeutic concepts will improve the clinical outcome in patients with CBF leukemia and activated RTK mutations. Neoangiogenesis plays an important pathophysiological role in the growth of hematological malignancies such as AML [1, 2] . Increased numbers microvessels have been detected on histological sections of bone marrow biopsies of AML patients as compared to those with reactive disorders. Besides increased provision of oxygen and nutritions, paracrine exchange of growth factors between AML blasts and endothelial cells is believed to contribute to the progression of AML [3] . The angiogenic growth factors are divided into endothelial specific and nonspecific factors. To the last class belong cytokines such as FGF or hepatocyte growth factors which have pleiotropic actions on various cell types. The VEGF family and the angiopoietins on the other hand are endothelial specific since their receptors are almost exclusively expressed on endothelial and immature hematopoietic cells [4, 5] . Both classes of receptors belong to the tyrosine kinase family [6] [7] [8] [9] . Interactions between members of the VEGF and angiopoietin family are necessary for efficient growth and remodeling of blood vessels [10] [11] [12] .

Our group investigated m-RNA expression of VEGF-A, VEGF-C, angiopoietin-1 and -2 in 90 AML patients younger than 61 years at diagnosis who were entered into the multi-center AML SHG Hannover study 13. In univariate analysis of overall survival of various clinical parameters and m-RNA expression levels of angiogentic cytokines, only a blast count <5% in a hypoplastic marrow on day 15 after start of induction chemotherapy, karyotype and expression of angiopoietin-2 reached statistical significance. On multivariate analysis of these variables, only a blast count <5% and angiopoietin-2 expression were identified as prognostic factors for overall survival. When the patients were seperated into groups of high vs. low expression of VEGF-A, VEGF-C or angiopoietin-1, the positive impact on overall survival of angiopoietin-2 expression was most pronounced in the groups with low expression of angiogeneic factors. These findings imply that high angiopoietin-2 expression may lead to blockade of the angiopoietin receptor tie-2 and in concert with low VEGF expression to a reduced angiogenic potential. In support of our data, in several experimental models, angiopoietin-2 led, in the absence of VEGF-A, to endothelial cell apoptosis and vessel regression [10] [11] [12] . These results suggest that simultaneous inhibition of VEGF and angiopoietin-1 effects may result in superior leukemic control.

To further substantiate our concept, we developed a SCID mouse AML model using M1 murine leukemia cells. Delivery of recombinant protein can be achieved by microencapsulation technology using stably transfected [14, 15] . Cells have been engineered for production of Ang2 and sNeuropilin-1 (sNP-1), a soluble VEGF receptor acting as a VEGF antagonist. The experiments has been set up to examine the effect of angiopoietin-2 or sNP-1 alone or in combination of both factors to test whether inhibition of tie2 in absence of VEGF activity might inhibit both leukemic cell proliferation and angiogenesis in vivo.

In clinical trials, blockade of VEGF action has been studied in several smaller phase II studies. SU5416 has been tested in 42 patients with refractory AML16. Partial remissions have been observed in 8 patients. Due to the twice weekly administration of SU5416 therapeutic plasma levels could be achieved only temporarily resulting in insufficient receptor blockade. Nethertheless patients with high initial VEGF expression had better response rates than those with low VEGF-mRNA levels supporting the concept of anti-angiogeneic therapy. In a recent study 15 patients with refractory AML were treated in a phase 1 study with SU11248, an oral kinase inhibitor of Flt3, Kit, VEGF and PDGF receptors17. Six patients had partial responses. Especially patients with FLT3 mutation had a higher response rate and response duration than patients with wildtype FLT3. Reductions of cellularity and numbers of Ki-67, phospho-Kit, phospho-KDR, phospho-STAT5 and phospho-Akt positive cells were detected in bone marrow histology analysis indicating target inhibition by SU11248. Recently, treatment results with CEP-701, a combined inhibitor of VEGF receptors and FLT3, of 14 patients with refractory AML with mutated FLT3 have been published18. Five patients had reductions of blood and bone marrow blasts. One combination trial of chemotherapy with bevacizumab, the humanized VEGF antibody, has been published 19. Forty-eight patients with relapsed or refractory AML were treated. The complete response rate was 33% which compared favorable with a historic control group.

In summary, inhibition of neoangiogenesis in AML by blockade of the VEGF pathway has shown some clinical efficacy. But treatment strategies have to be further refined. Pregnancy leads to a mutual flow of cells between mother and child. The exposure to the either foreign maternal or to foreign paternal minor H antigens during pregnancy drives the generation of minor H antigen specific T cells in mutual direction. We study the relevance of the minor H antigen immune responses and their immune regulation in relation to subsequent HSCT or Cord Blood (CB) transplantation.

In the HLA matched minor H antigen HA-1 mismatched setting of renal transplantation, an HA-1 specific allo-immune response leading to renal allograft tolerance has been analyzed. One of the patients had discontinued immunosuppression over 30 years ago while sustaining normal kidney function. HA-1 specific T regulator cells were present in the latter long-term kidney allografted recipient. These cells coincided with HA-1 specific CTLs and HA-1 microchimerism. Herewith a novel characteristic for human minor H antigens has been disclosed with potential impact for decrement of immunosuppressive therapy in solid organ grafting.

AML in Children. Experiences from the NOPHO Studies Common Nordic AML protocols have been in use since 1984. The studies have documented the feasibility and effectiveness of high-dose cytarabine based consolidation. The induction regimen in NOPHO-AML88 was very intensive resulting in less relapses but too many toxic deaths. In NOPHO-AML93 up-front intensity was reduced in good responders by postponing the second induction course until haematological recovery resulting in both reduced toxicity and improved outcome. The most important prognostic factors were response to first induction and cytogenetics. Patients with t(9;11), t(8;21) and inv(16) had a favourable outcome as compared with a poor outcome in 11q23 rearrangements other than t(9;11). The 5-year event-free survival, disease free survival and overall survival in NOPHO-AML 93 was 50%, 54%, and 66%, respectively.

Down syndrome (DS) is found in approximately 15% of the NOPHO-AML cases. Myeloid leukaemia in DS has many unique features including a remarkably good prognosis. Myeloid leukaemia of DS treated on NOPHO-AML88 and -93 showed improved outcome with reduction in treatment dose as well as less intensively timed treatment compared with standard AML regimens. The current NOPHO-AML 2004 has adopted a risk-adapted therapy. High-risk patients are defined by the presence of 11q23 aberrations other than t(9;11) or poor response with more than 15% blasts in the BM on day 15. Only high-risk patients will be offered SCT and the study will compare the outcome following the genetic randomisation between HLA-matched sibling donor and HLAmatched unrelated donor. The effect of addition of two courses of Gemtuzumab ozogamicin (Mylotarg) to standard consolidation therapy will be evaluated in a randomised setting in standard risk patients. The trial will also evaluate the prognostic value or MRD-status at fixed time points during treatment.

NOPHO-AML84, -88, and -93 Following the introduction of intensive anthracycline and cytarabine based AML therapy in the 1970's a pilot study, started in Oslo in 1981, using a modified induction therapy from the MRC group with the combination of doxorubicin, cytarabine, and 6-thioguanine. The study introduced consolidation therapy in childhood AML with high-dose cytarabine, 2000 mg/m2 twice daily for 3 days repeated four times. Maintenance consisted of monthly courses of cytarabine and 6-thioguanine for one year. The results were promising, with the first 8 patients remaining in remission 5-29 months after diagnosis [1] .

The first common Nordic protocol for AML was opened July 1, 1984 (NOPHO-AML84). The results of NOPHO-AML84 formed the basis for the following protocol (NOPHO-AML88) [2] . Compared to other international series, the frequency of resistant disease (15/105) was too high in NOPHO-AML84 and many patients experienced relapse (45/82). The induction therapy of NOPHO-AML88 was intensified by giving three induction courses. The first and third course consisted of continuous infusion of cytarabine and etoposide plus 6thioguanine and doxorubicin. The second course consisted of mitoxantrone and continuous infusion of cytarabine. The interval between the first and second course was intended to be as short as possible. The consolidation was intensified by adding mitoxantrone and etoposide to alternate courses of highdose cytarabine.

NOPHO-AML88 showed a non-significant trend of improving EFS (42% vs. 32%) compared with NOPHO-AML84 [2] . However, the toxicity of NOPHO-AML88 was not acceptable, with 14/118 dying in aplasia and 10 of 58 dying in CCR.

The NOPHO-AML93 study used the same therapeutic blocks as in NOPHO-AML88, but the approach changed [3] . After the first course the patients were observed until BM showed persistent disease or CR. Those who obtained CR (67%) after the first course were given a second identical course. Patients with persistent disease received mitoxantrone and cytarabine as the second course. Only two courses of induction were given. The consolidation therapy remained the same as in NOPHO-AML88.

By December 2001, 243 children without Down syndrome had been enrolled on NOPHO-AML93. Analysing extended follow-up data the 5-year EFS increased from 44% in the -88 protocol to 50% in the -93 protocol. Toxic death during induction was reduced to 2% and 92% achieved remission. The 5-year OS increased from 50% in NOPHO-AML88 to 66% in NOPHO-AML93 [5] . The main prognostic factor in NOPHO-AML93 was the in vivo response to the first course of therapy. For those achieving remission after one course (67%), the EFS was 56% compared with 35% in those not in remission after first induction (figure 1).

Informative cytogenetics was obtained in 91% of the patients in NOPHO-AML93 [3;4] . Patients with t(9;11)(p22;q23) had significantly better EFS (86%) than other cytogenetic groups, and t(8;21) and inv (16) carried an intermediate prognosis (Table 1) . A very poor prognosis was found in patients with 11q23 aberrations other than t(9;11) and in a small group of patients with a highhyperdiploid karyotype.

EFS was superior in those receiving SCT in first complete remission (CR1), p = 0.04, but the OS did not differ between SCT and chemotherapy only in CR1 ( Figure 2 ).

The max cumulative dose of anthracycline in NOPHO-AML 93 was 375 mg/ m2. 

The current trial opened in January 2004. Idarubicin replaced doxorubicin during induction. Risk-adapted therapy was introduced. High-risk patients are defined by the presence of 11q23 aberrations other than t(9;11) or poor response defined as more than 15% blasts in the BM on day 15 from start of induction. Providing a matched related or unrelated donor is available all high-risk patients will receive SCT following at least one consolidation course. The study will compare the outcome of SCT following the genetic randomisation between HLA-matched sibling donor and HLA-matched unrelated donor. Standard risk patients and high-risk patients without a donor are given four courses of highdose cytarabine based consolidation. Following consolidation the patients are randomised to receive two courses of Gemtuzumab ozogamicin (GO) 5 mg/m2 or no further therapy. The design of the study aims at evaluating the role of GO as post consolidation therapy of minimal residual disease. The trial will evaluate the prognostic value or MRD-status at fixed time points during treatment.

The Nordic studies have documented that children with DS represent a large subgroup in AML. In our population-based material we found that children with DS constituted almost 15% of the AML cases [2;6] . NOPHO was among the first to show that children with DS have a very special type of AML with a remarkably good prognosis [7] . Myeloid leukaemia in children with DS is now considered a separate entity [8] . An analysis of 56 children with DS treated on the NOPHO-AML88 and '93 protocols showed that in the dose-intensive NOPHO-AML88 protocol 8 out of 15 patients (53%) experienced an event. In the less dose-intensive NOPHO-AML93 protocol 7 out of 41 patients (17%) had an event [9] . Therapy was reduced in 29 patients (52%) with in average 75% and 67% of the scheduled dose of anthracycline and cytarabine, respectively. Treatmentrelated death occurred in seven all of whom had received full treatment doses. Relapse and resistant disease occurred at a similar rate in those receiving full and reduced treatment. A reduction in treatment dose as well as a less intensively timed treatment compared with standard AML regimens seems to improve the outcome for patients with DS and myeloid leukaemia [9] . The current NOPHO-AML 2004 protocol recommends reducing all anthracycline doses in DS to 67% of the doses in non-DS patients and advocate adequate clinical and haematological recovery between treatment courses. Abstract: Genetic and molecular techniques have provided increasing insights into the biology of acute myeloid leukemia (AML). These investigations showed that AML is not a homogeneous disease but a heterogeneous group of biologically different subentities. These subentities are currently primarily defined by cytogenetics by which three main subgroups can be discriminated: AML with balanced translocations, AML with unbalanced aberrations and AML without cytogenetically detectable aberrations. Within the latter group molecular alterations are identified in more than half of cases such as NPM mutations, FLT3 mutations, MLL duplications and mutations of CEBP-. The clinical meaning of these findings is illustrated by substantial differences in response to therapy and long term outcome. As demonstrated by the recent multicenter trial of the German AML Cooperative Group (AMLCG) and other studies intensification of induction therapy may improve the results in distinct subtypes but fails to do so in others. Therefore, new strategies need to be explored which incorporate the knowledge about the biology of AML to develop biology adapted treatment strategies.

Acute myeloid leukemia (AML) is a heterogeneous group of disorders that arises from the malignant transformation of early hematopoietic precursor cells. Modern diagnostic techniques allow to unravel the degree of heterogeneity in detail. (14, (23) (24) (25) . By cytogenetics two major groups of AML can be discriminated: One group with chromosomal aberrations accounting for approximately 52 % of all de novo AML and a second group without detectable karyotype abnormalities. Within the first group two major subtypes can be further distinguished. One comprises AML with balanced aberrations mainly consisting of t(8;21), t(15;17) and inv 16. The second group includes cases with unbalanced aberrations such as 5q-,7q-, -5, -7 and complex karyotypes. In AML without detectable cytogenetic abnormalities molecular analyses reveal distinct molecular aberrations such as mutations of FLT-3, CEBP-or KIT and partial tandem duplications of MLL (6, 16, 21) . Most recently mutations of the nucleophosmin (NPM) gene were described to occur in more than half of patients with a normal karyotype (4).

The discrimination of cytogenetic subgroups is of high clinical relevance. Hence, AML with balanced aberrations have a good prognosis with long term survival and potential cure in approximately 60% -80% of cases. AML with nonbalanced aberrations on the other hand have a poor outcome with only 10% -15% long-term survivors. AML with no detectable abnormalities or other cytogenetic aberrations comprise a group with an intermediate prognosis in which long term survival is achieved in approximately 25% -30% of cases 1, 3, 6-8).

The overall prognosis of patients suffering from AML has steadily improved over the last three decades. Nowadays, complete remissions are achieved in 60 -70% of all patients with long term disease free survival and potential cure in 25 -40% of cases. A more detailed analysis indicates that this progress has mainly been achieved in patients <60 years of age while in older patients little improvements have been obtained (2, 12, 13) . This conclusion must take into account, however, that the proportion of patients at higher age that is included into intensive treatment protocols has steadily increased over time (Tbl. 1). Hence, it may be speculated that the overall survival of elderly patients with AML has possibly increased which cannot be proven because no information is available about the outcome of corresponding cases who have been treated outside clinical trials or who have previously not been treated at all.

When analysing the approaches that underly the progress in AML therapy two major developments appear essential: the intensification of therapy and the improvement of supportive care. The latter includes the effective substitution of blood components and of thrombocytes through platelet transfusions in particular, as well as active antimicrobial agents directed against bacterial and fungal infections. These measures provide the basis for more intensive therapies that are associated with severe and long lasting bone marrow aplasia. This global overview indicates that the progress in AML that has been achieved during the last few decades was mainly based on the escalation of AraC dose and the development of double induction therapy. This conclusion also reflects the fact that since the introduction of AraC and DNR into leukema therapy in the mid 60ies no effective new agent has become available so that clincians were forced to concentrate their efforts on the improved application of "old" drugs.

The hope that the still unsatisfactory prognosis of patients suffering from AML may change in the near furture is mainly based on two recent developments: increasing insights into the biology and pathogenesis of the disease and the development of new and effective antileukemic agents.

The increasing insights into the biology and pathogenesis of AML as gained by cytogenetic and molecular techniques allow to discriminate distinct subgroups of AML with a different clinical outcome. Besides the cytogenetic discrimination between karyotypes with balanced, unbalanced or no detectable chromosomal aberrations further prognostically relevant subtypes can be identified. Hence, within the group of AML with unbalanced chromosomal aberrations the subgroup with complex aberrant karyotypes has a significanly poorer outcome than patients with unbalanced, non-complex karyotypes (1, 15, 20, 22) . Within the group of AML without cytogenetically detectable chromosomal aberrations gene mutations such as FLT3 length mutations, MLL partial tandem duplications or mutations of C/EBPalpha can be identified and are of prognostic relevance (5, 17, 18) . Recently, NPM mutations were shown to predict a favorable outcome in patients with normal karyotype (19) .

In addition, new markers for in vivo chemosensitivity or -resistance, respectively, have recently been described. Based on the data of the AMLCG studies Kern et al. reported that the degree of blast cell reduction in the bone marrow on day 16 of therapy was closely related to long term outcome (12) . Based on these parameters five prognostically relevant subgroups of AML can be defined (Tbl. 2) (9).

So far, these analyses allow to define distinct subgroups of AML with different prognoses. They are describing the current situation at a more precise level. The main challenge for clinicians, however, remains the question of how to treat these different subgroups and how to improve on their current outcome. Today, no firm data exist that allow to determine the best treatment for any of these subgroups. Therefore, an analysis of treatment outcome for distinct subgroups is necessary which is based on an unbiased global protocol. Such an approach was taken by the most recent study of the AMLCG that included adult AML patients >18 years of age without an upper age limit. Except for AML with t (15;17) that were treated in a separate protocol, all AML cases were eligible for the study including de novo AML, secondary AML, AML deriving from a preceding hematologic disorder such as a myelodysplastic syndrome (MDS) and high risk MDS cases. The treatment concept included three major questions which were addressed in way of prospective randomized comparisons (Fig. 1) .

The main therapeutic questions comprised (1) the comparison of double induction with Thioguanine, Daunorubicin and conventional dose AraC (TAD 9) followed by high-dose AraC plus Mitoxantrone (HAM) as second induction cycle versus the HAM -HAM sequence; (2) the comparison of priming with G-CSF versus no G-CSF and (3) the comparison of consolidation by myeloablative therapy followed by autologous transplantation versus conventional consolidation by TAD 9 followed by three years monthly maintenance. All three randomizations were stratified for age > or < 60 years of age, the type of AML, cytogenetics and the pretreatment level of serum LDH. With more than 2000 patients that were recruited into this trial between 1999 and 2004 no significant differences between the TAD 9 -HAM versus the HAM -HAM sequence are currently observed for remission rate, remission duration or overall survival. This may either reflect a comparable antileukemic activity of both regimens or may indicate that such a gross and superficial analysis is not adequate to identify different outcomes within distinct subentities. Table 3 reveals that the afore mentioned five different subgroups still are of prognostic relevance under the conditions of the AMLCG 1999 study. A comparison of the two induction regimens within these subgroups, however, suggests a higher activity of the HAM -HAM sequence particularly in patients with nonbalanced, non-complex karyotypes. In patients with CBF leukemias no differences between TAD 9 -HAM versus HAM -HAM are observed either. A detailed evaluation of the two major subgroups, however, shows different response patterns. Hence, the TAD 9 -HAM sequence seems superior to HAM -HAM in cases with t(8;21) whereas an opposite relation is seen in cases with inv(16) or t(16;16) (Tbl. 4).

These few examples indicate that therapy and biology are not independent in determining prognosis but that a biology oriented therapeutic approach may have a substantial impact on treatment outcome.

The limitations that prohibit to follow this direction with greater speed at the present time are set by the small number of currently available antileukemic agents.The development of AML therapy is therefore determined by the progress of basic science but even more by the translation of this knowledge into the development of new agents by pharmaceutical companies. The steps that can Despite a large body of data supporting the rationale for combining chemotherapy for acute myeloid leukemia (AML) with agents which inhibit the function of the ATP-binding cassette transmembrane transporter P-glycoprotein (Pgp) [1] outcomes of phase 3 trials conducted in older previously treated [2] and untreated [3, 4] patients, have been disappointing Even so, fresh laboratory data are emerging which continue to underscore the importance of Pgp function in conferring drug resistance on AML cells isolated from patients undergoing uniform treatment regimens [5] .

Within the Cancer and Leukemia Group B (CALGB), four trials between 1994 and 2003 have studied the toxicity and efficacy of the cyclosporin A (CsA) analogue PSC-833 (PSC, Valspodar) when used during induction chemotherapy in younger [6, 7] and older (>60 years) [3, 8] patients with untreated, de novo AML.

The trials are outlined in Table 1 . All patients received identical therapy with Ara-C (A), given by c.i.v. at doses of 100 mg/m2 daily for 7 days. Daunorubicin (D) and etoposide (E) were each given by short i.v. infusion on days 1-3. E was added to the traditional "7 and 3" A and D induction framework in order to take advantage of the potential for Pgp inhibition with PSC to increase the intracellular accumulation of two anti-leukemic agents. Residual leukemia was treated using "5 and 2" ADE or ADEP regimens using the same doses of A, D and E used during the first induction course. PSC was given as a continuous i.v. (c.i.v.) infusion in ADEP, overlapping the periods during which D and E were given.

Dose finding studies in older (CALGB 9420) and younger (CALGB 9621) patients were conducted which established induction regimens, with (ADEP) and without (ADE) PSC, which were thought to be reasonably equivalent with respect to toxicity and clinical efficacy. The dose ranges of daunorubicin and etoposide evaluated in the phase 1 trials, and the doses chosen for phase 3 testing, are presented in Table 1 . It was recognized that Pgp inhibition would affect the metabolism and clearance of daunorubicin and etoposide. Furthermore, the potential existed for mutual interactions between the two Pgp substrates and the transport pump. Ultimately, the doses chosen for combination therapy with PSC, which were based on the attainment of purely clinical parameters in the form of dose-limiting toxicities (DLT), reflected what might have been expected from measurements of drug serum levels and clearance.

In the older patient population, a relatively high level of precision was attained with respect to defining equitoxic doses of daunorubicin and etoposide, with and without PSC, by means of treating a greatly expanded number of patients in each cohort during the dose-escalation portion of the trial. This represented a departure from traditional phase 1 trial designs in which the reliability of the clinical findings is constrained by the small number of patients studied. Among younger patients, only a rough estimation of equitoxicity in the control and experimental regimens was established for phase 3 testing. Substantial mucosal and generally reversible hepatotoxicty rapidly placed limits on ADEP escalation while, unexpectedly, DLT as strictly defined proved elusive to attain in younger patients even with daunorubicin doses of 95 mg/m2 and etoposide doses of 150 mg/m2 in the absence of PSC. The doses of daunorubicin and etoposide used in the phase 3 trial therefore reflected considerations regarding observed and potential clinical efficacy and toxicity based on our analysis of outcomes among the 410 patients who were treated in the phase 1 trial. Based on pharmacologic considerations, the dose reductions of daunorubicin (56%) and etoposide (60%) in ADEP relative to ADE likely lead to similar serum levels of the Pgp substrates in the 2 treatment arms in the phase 3 trial. Cerebellar toxicity attributable to PSC and cardiotoxicity from the high doses of daunorubicin used in ADE have not been significant. The phase 3 trial in older patients was halted after 120 patients were randomized because of toxicity concerns, principally early death in the ADEP arm. Despite this, overall survival (OS) did not differ by treatment arm. There was also a suggestion that patients whose leukemic cells displayed PSCmediated drug efflux in vitro and received ADEP had a clinical benefit with respect to disease-free survival (DFS) as compared with those patients treated with ADE (5 vs. 14 months, p=0.07) [3] .

Encouraging findings with respect to prolongation of DFS and OS among patients < 45 years treated on CALGB 9621 stimulated interest in pursuing the phase 3 trial in patients < 60 years. After 302 patients had been randomized, PSC became unavailable for clinical use, ending the randomized induction portion of that trial, CALGB 19808. With a median follow up of 3 years and a minimum follow up of greater than one year since the last patient was treated, OS and DFS results of the randomized induction are presented in Table 2 , alongside the corresponding outcomes observed in the phase 1 trial. The overall complete remission incidence was identical for ADE (77%) and ADEP (78%). Detailed analysis of the induction results of the randomized trial is being undertaken. Factors to be reviewed include the distribution of cytogenetic risk groups between the treatment arms, the impact of allografting in CR, the fact that patients with minimal evidence of prior myelodysplasia were included in the phase 3 but not in the phase 1 study, and the possible influence of lower doses of daunorubicin and etoposide used in ADEP in the phase 3 study as compared with the median doses used in the group of 410 patients participating in the phase 1 trial. Furthermore, the impact of the lower statistical power resulting from the premature ending of the induction randomization, which was terminated with about half of the intended number of patients evaluable, has to be analyzed.

Notwithstanding the disappointing clinical outcomes observed to date, the strength of the association between Pgp expression and outcomes in AML as measured by immunohistochemical and functional techniques continues to drive ongoing explorations of strategies aimed at inhibiting Pgp function in conjunction with chemotherapy in AML. Data favoring use of Pgp inhibition using CsA among patients with relapsed and refractory AML [9] have lead to an ongoing phase 3 trial being conducted by the Southwest Oncology Group in untreated patients. Zosuquidar trihydrochloride, a Pgp inhibitor without pharmacokinetic interactions with Pgp substrates [10] , is undergoing evaluation by the Eastern Cooperative Oncology Group in a phase 3 trial in combination with daunorubicin and cytarabine, also in untreated patients. Both trials are confined to patients over age 55. A hypothesis that remains to be refuted is that the clinical benefit of Pgp modulation may be more discernable in younger patients with AML, whose leukemia cells are less likely to harbor multiple mechanisms of drug resistance. Coexpression of Pgp and MRP, for example, increases with age as well as with the adverse cytogenetic findings, which are more prevalent in older patients [11] . Given that leukemic stem cells may harbor a reservoir of Pgp-expressing [7] progenitor cells [12] , a basis exists for continued exploration of means to effectively and safely inhibit Pgp function in younger patients receiving chemotherapy. Daunorubicin and Etoposide were each given daily by short i.v. infusion on days 1-3, along with Ara-C 100 mg/m2 by c.i.v. on days 1-7. PSC 2.8 mg/kg loading dose was given i.v. over 2 hours prior to start of chemotherapy, followed by 10 mg/kg. c.i.v. for 72 hours on days 1-3.

Acute lymphoblastic leukemia (ALL) is rare in adults, and available data on ALL in older patients are relatively scarce. One population based registry reported that 31% of all adult cases occurred in patients 60 years or older. The success that has been achieved treating children and younger adults with acute lymphoblastic leukemia has not been paralleled in older adults. Likely reasons include the presence of coexisting medical disorders leading to decreased tolerance for chemotherapy as well as fundamental biological differences in the spectrum of ALL seen in this population. There is considerable evidence that the clinical and biological characteristics of the disease changes dramatically between childhood, the middle adult years, and older age groups. Pretreatment clinical data from 909 adults enrolled on multicenter clinical trials by the Cancer and Leukemia Group B (CALGB) between 1988 and 2005 are shown by age cohort in Table 1 . Significant agerelated differences were also noted in the immunophenotype and karyotype results when 69 patients between 60-89 years old (median, 68) were evaluated at the Edouard Herriot Hospital in Lyon, France between 1980 and 1998, and were compared with 309 younger adults 15-59 years old (median, 29) [1] . Lymphoblasts were of B-cell lineage in 89% of the older patients compared with 66% of the younger adults (p=0.0004). Conversely, T-cell ALL was present in only 8% of older patients but 29% of the younger patients (p=0.0007). Myeloid antigens were co-expressed by 19% of the older adult cases compared with 11% of the younger cases; lymphoblasts from 69% of older ALL patients also expressed CD34.

A similar analysis has been reported by the German Multicenter ALL Study Group (GMALL), although the older age group was truncated at 65 years old. [2] Between 1984 and 1999, 342 patients (12%) from 55-65 years old were enrolled on one of 4 prospective clinical trials and were compared with 2463 patients (88%) ages 15-54 years. The older patients less frequently had peripheral lymph node involvement, mediastinal tumor, or splenomegaly. A common or pre-B immunophenotype was more frequent among older patients (75% vs 59%; p=0.001) whereas T-cell ALL was less frequent (3% vs 14%), and the frequencies of null plus pro-B ALL were similar (13% vs 12%). Among T-ALL cases, mature T-ALL (2% vs 7%) and thymic T-ALL (3% vs 14%) occurred less commonly in older patients. In a recent multicenter series of older ALL patients from Poland, no remarkable differences in pretreatment characteristics were reported between 64 patients 60-69 years old and 23 patients 70 years and older [3] . Cytogenetic abnormalities characterize the biological heterogeneity of ALL and provide a strong and independent prognostic factor for treatment outcome. The incidence of Philadelphia (Ph) chromosome positive ALL, a subset that is resistant to conventional chemotherapy, clearly increases with age [4] . Clonal chromosomal abnormalities were found at diagnosis in 378 of 443 adults (85%) by the Groupe Francais de Cytogenetique Hematologique; 60 (13%) were older than 60 years (range, 60-84) [5] . In this series, 18% of those less than 40 years old and 46% of those 40-60 years old were Ph+ compared with 35% of those older than 60 years. Among 276 patients (ages 16-83) with centrally reviewed karyotypes enrolled on a prospective study by the CALGB, 76% had a clonal abnormality; 28% overall and 33% of those older than 60 years had a t(9;22) [6] . In a recent report from the Southwest Oncology Group, the frequency of Ph+ ALL increased from 6% in those less than 25 years old to 14% for those 25-35 years old, 33% for those 36-55 years old, and 53% for patients older than 56 years [7] . In the large GMALL series described above, among those with common or pre-B ALL, the incidence of Ph+ or BCR/ABL+ ALL was significantly higher among adults older than 55 years than younger adults (54% vs 37%; p=0.001) [2] . In the French series from Lyon, the Ph chromosome or BCR/ABL fusion gene was detected in 24% of older patients and 19% of young patients [1] .

With a few exceptions, older patients with ALL have usually been excluded from clinical trials by eligibility criteria. Regimens developed for younger adults have been truncated at 60 or 65 years old. For the past 2 decades, the CALGB has not had an upper age limit for enrollment onto its frontline ALL treatment studies. Approximately 18% of clinical trial subjects have been 60 years or older (median, 66 years; range 60-83 years). Similarly, investigators at the M. D Anderson Cancer Center in Houston, Texas have actively enrolled older patients onto their frontline ALL studies [8] . The fraction older than 60 years has been 22%. In both this multi-center example and a single center experience, the proportion of older patients enrolled appears less than the proportion in the community.

In general, older cancer patients have been underrepresented in prospective clinical trials, and this can have serious consequences on the development of better therapies for these patients. This is particularly notable for patients greater than 75 years old. Although it is estimated that 54% of patients with leukemia in the United States are greater than 65 years old, only 24% of subjects enrolled on new drug trials for leukemia filed with the Food and Drug Administration (FDA) are older than 65 years [9] . In the United Kingdom, where on average 68 patients 60 years or older develop ALL every year, only 3-8 patients (4-15%) were enrolled each year on the national MRC UKALL X and XA studies. [10] Very few clinical trials have focused on therapies designed specifically for older patients with ALL, and fewer still have sufficient numbers to deal with the clinical and biological heterogeneity within this age group. Even when treated on the same trial as younger adults, older patients often receive attenuated doses of myelosuppressive drugs [11, 12] . Table 2 demonstrates the effect of increasing age on the outcomes of 759 adults with newly diagnosed ALL treated on 5 sequential CALGB studies between 1988 and 2002. The complete remission (CR) rate declined from 90% for those under age 30, to 81% between 30-59 years old, and to 57% for those 60 years and older. The overall survival at 3 years was estimated to be 58% (95% confidence limits, 52-64%) for those less than 30 years old, 38% (33-43%) for those 30-59 years old, and only 12% (7-19%) for the 129 patients older than 60 years. Similar results have been reported on by other cooperative groups and large leukemia centers.

The GMALL Study Group reported on a pilot study designed for patients older than 65 years. [13] Favorable factors for achievement of CR were pro-B ALL or T-ALL (69% CR compared with 36% for pre-B ALL), no Ph chromosome (64% vs 19% for Ph+), and WBC count 61 years received imatinib at 800 mg/day in combination with prednisone for 30 days, followed by imatinib alone as postremission treatment; 92% of patients achieved a CR. [24] The GMALL evaluated imatinib monotherapy in older Ph+ ALL patients in a prospective, randomized trial [25] . Patients with a median age of 67 years were randomly assigned to receive a 4 week course of imatinib at 600 mg/day or standard induction chemotherapy. After achieving a CR or partial remission (PR), all patients received imatinib concurrently with consolidation chemotherapy. Imatinib induction was significantly superior to chemotherapy alone, with 93% achieving a CR and 7% a PR. In contrast, 46% of evaluable patients allocated to the chemotherapy arm failed to enter remission. Severe infections were less frequent in patients allocated to imatinib induction, and there was no induction mortality [25] .

It is not likely that escalating the doses of currently available chemotherapy agents or altering their sequence of administration will markedly improve the outcome of treatment for ALL in adults. New drugs are needed. Because a large fraction of older patients have Ph+ ALL, major advances in the treatment of BCR/ ABL+ leukemia offer hope for considerable improvement. Second generation ABL kinase inhibitors such as AMN107 (Novartis) and dasatinib (BMS354825; Bristol Myers Squibb) are now completing phase II trials in patients with imatinib-resistant Ph+ ALL. If successful, these agents should be evaluated next in frontline therapy and in combination with chemotherapy.

Other targeted therapies currently being evaluated in ALL include monoclonal antibodies. Rituximab binds to CD20 which is expressed on more than 20% of lymphoblasts in about half of common or pre-B ALL cases. In a pilot study by the GMALL, when rituximab was given before each cycle of chemotherapy, the CR rate was 63% and survival after one year was 54% for 19 CD20+ patients older than 55 years (median, 66) [14] . Drugs with proven efficacy against ALL, such as vincristine and daunorubicin, have been modified as liposomal sphingomyelin/ cholesterol preparations, and are in clinical trials. Italian investigators have reported on a small trial using liposomal daunorubicin with vincristine and dexamethasone in 15 newly diagnosed older ALL patients (median age, 69 years) [26] . The CR rate was 73%, the median disease-free survival (DFS) was 21 months, and the 2-year DFS was 36%. It is possible that some combination of these newer drugs will overcome the innate resistance present in older patients with ALL, and at the same time improve the overall tolerance to treatment. Intensive chemotherapy is essential to stabilize complete remission induced by all-trans retinoic acid (ATRA), and to achieve cure in patients with newly diagnosed acute promyelocytic leukemia (APL). This observation suggests that the antileukemic efficacy of the chemotherapy that is combined with ATRA has influence on the outcome. In the treatment protocol of the AMLCG for newly diagnosed APL started in 1994, the induction chemotherapy combined with ATRA is intensified by the incorporation of high dose cytosine arabinoside (ara-C). The therapy consists of TAD/HAM double induction and ATRA followed by TAD consolidation and three years maintenance chemotherapy. In patients aged over 60 years, the intensity of the chemotherapy is reduced. The second induction cycle (HAM with reduced ara-C dose) is only given in the case of insufficient response to the first induction cycle. The AMLCG strategy proved highly effective in all risk groups of APL.

In order to investigate which APL patients benefit most from this treatment approach, in the new APL protocol of the AMLCG (APL 2005 protocol) patients are randomly assigned to the AMLCG strategy and to the concept of the Spanish PETHEMA consisting of ATRA and idarubicin/ mitoxantrone. Patients with a molecular or hematological relapse qualify for treatment with ATO within a European relapse protocol, which has been started by the AMLCG in January 2005.

Intensive chemotherapy is essential to stabilize complete remission induced by ATRA, and to achieve cure in patients with newly diagnosed APL. This observation suggests that the antileukemic efficacy of the chemotherapy that is combined with ATRA has influence on the outcome.

The concept of the German AML Cooperative Group (AMLCG) for treatment of newly diagnosed APL is based on the results of the randomized AML-86 protocol for all subgroups of acute myeloid leukemia (AML) including APL. In this protocol, the treatment consists of a double induction therapy with ara-C in standard dose (TAD/TAD), or with high dose ara-C (TAD/HAM), followed by TAD consolidation and three years monthly maintenance chemotherapy [1] . This strategy proved highly effective in the subgroup of patients with APL. The nine year relapse free survival rate of APL patients was 47%, with a significant benefit of the high dose ara-C arm (70% vs. 19%; p=0.02) [2] .

After the introduction of ATRA, the prospective multicenter study for patients with newly diagnosed APL (from December 1994 to October 2005) was initiated with the intention to combine the positive effects of the differentiating substance ATRA with a chemotherapy strategy having a high curative potential. Therefore, the induction therapy consists of ATRA and double induction chemotherapy (TAD/HAM), intensified by high dose ara-C. This is followed by consolidation and maintenance chemotherapy. Monitoring of minimal residual disease is performed by RT-PCR of PML/RAR transcripts in order to assess quality and stability of remission on the molecular level.

APL 1994 protocol of the German AMLCG Induction therapy consists of TAD/HAM double induction chemotherapy and of the simultaneous administration of 45 mg/m ATRA daily until complete hematological remission or for a maximum of 90 days. The HAM course is given on day 21 after the start of TAD independent on cytopenia. Two to four weeks after CR, one consolidation course of TAD is given followed by three years cyclic monthly maintenance chemotherapy (Figure 1 ). Patients older than 60 years receive a less intensive induction chemotherapy: The second induction cycle (HAM with reduced ara-C dose of 1 g/m2 instead of 3 g/m2) is only given, if complete remission is not achieved after the first cycle (Table 1) . Molecular monitoring of PML/RAR is scheduled after induction, after consolidation and every three months during maintenance therapy. In the case of persistence or reappearance of PCR-positivity for PML/RAR after consolidation or during maintenance, patients are to undergo allogeneic bone marrow transplantation (BMT), if a related donor is available. Patients not eligible for BMT receive 45 mg/m ATRA over 7 days in addition to the chemotherapy in each maintenance course.

In the present APL study, the induction chemotherapy in combination with ATRA is intensified by the incorporation of high dose ara-C and by the early application of a second induction cycle after an interval of 21 days (double induction) independent on the recovery of blood counts. The patients' data were routinely updated every half year and presented at the meetings of the German AMLCG since the start of the APL study in 1994. The summarized results of earlier reports of the present study indicate the high antileukemic efficacy of high dose ara-C and its importance in newly diagnosed APL [2] [3] [4] [5] : • With the AMLCG strategy including high dose ara-C and ATRA during induction therapy a low relapse rate in all 'risk groups' of APL was seen. • The initial WBC count was no statistically significant risk factor for relapse. • Poor prognosis might be improved by the rapid reduction of the malignant clone (molecular remission in 90% after 6 to 8 weeks).

• No CNS relapses were observed in the present AMLCG study so far. • High dose ara-C in APL proved highly effective in the pre-ATRA era (AML-86: RFS 70% vs. 19%, p=0.02). The results of the last update (August 2005), including the data of 166 evaluable patients with newly diagnosed APL confirm these observations after a median observation time of approximately five years.

The subsequent study (APL 2005 protocol of the German AMLCG) was started in November 2005. In this study, the previous concept of the German AMLCG (with slight modifications) and the concept of the Spanish PETHEMA group will be randomly compared (Figure 2) [7] . The assessment of the kinetics of the minimal residual disease after each step of induction and consolidation and during follow up by qualitative nested PCR and quantitative REAL-time PCR is mandatory. This randomized concept enables the comparison of both strategies in the different risk groups of APL.

Arsenic trioxide (ATO) is now regarded as the treatment of choice for relapsed APL [6, 8] . Against the background of the high toxicity of chemotherapy and the good tolerability of ATO with a high rate of efficacy, the question arises as to what extent the use of traditional chemotherapy or its intensification can be dispensed with if ATO is used. 

The presence of Philadelphia chromosome (Ph) with the formation of BCR-ABL gene rearrangement is known to be the most adverse prognostic factor for ALL, and occurs in up to 30% of adult ALL. Because long-term survival can hardly be achieved by conventional chemotherapy alone, there is a clear need for alternative treatments. Imatinib is a potent selective inhibitor of the BCR-ABL tyrosine kinase, and induced response in a substantial proportion of Ph-positive ALL (Ph+ALL), but the response was not durable [1, 2] . We recently reported our preliminary results of imatinib-combined regime for 24 newly diagnosed Ph+ ALL, producing 96% CR [3] . 18 Here, we present the results on 80 newly diagnosed Ph+ALL, comparing with those of our historical controls treated with chemotherapy alone.

The JALSG-ALL202 study has previously been described in detail [3] . Newly diagnosed BCR-ABL-positive ALL of age between 15 and 64 was registered after written informed consent. Pre-treatment bone marrow (BM) was subjected to the multiplex reverse transcription (RT)-PCR test, and patients positive for BCR-ABL were treated with the Ph regimen. For remission induction therapy, imatinib 600 mg daily was administered from day 8 to day 63 in combination with cyclophosphamide, daunorubicin, vincristine and prednisolone. Consolidation therapy consisted of an odd course comprising high-dose methotrexate (MTX) and high-dose cytarabine (Ara-C) and an even course with single-agent imatinib 600 mg daily for 28 days. Those were alternated for 4 cycles each. After the consolidation therapy, patients received maintenance therapy consisting of vincristine, prednisolone and imatinib 600 mg daily up to 2 years. Central nervous system (CNS) prophylaxis was done by intrathecal MTX, Ara-C and dexamethasone during the remission induction and each consolidation course (9 times in total). Allogeneic SCT was recommended if HLA-identical sibling donor was available, and SCT from an alternative donor was allowed. The protocol was reviewed and approved by the institutional review board. The numbers of BCR-ABL copies in BM samples was evaluated with the real-time quantitative RT-PCR (RQ-PCR) at diagnosis, on days 28 and 63, after the first and third cycles of consolidation therapy, after 1 year of treatment and at the end of the entire therapy in a central laboratory. The threshold for quantification was 50 copies/g RNA, which corresponded to a sensitivity of 10-5. The PCR negativity was categorized as the level below the threshold. The results for Ph+ALL cases in the JALSG ALL93 study [4] were used as historical controls.

Between September 2002 and January 2005, 80 patients with newly diagnosed BCR-ABL-positive ALL were entered and all were evaluable. There were 49 males and 31 females with a median age of 48 years (range, 15 to 63 years). Three patients had CNS leukemia at diagnosis. Median follow-up was 13.1 months (range, 2.2 to 35.3 months) for surviving patients. CR was achieved in 77 (96%). The median time to CR was 28 days (range, 19 to 69). Two early deaths occurred during the induction therapy, and one patient, for whom imatinib had to be discontinued due to ileus, did not reach CR. The CR rate was significantly higher than the 51% achieved for 51 patients treated with chemotherapy alone in the JALSG ALL93 study (p<0.0001) [4] . Severe toxicity associated with the remission induction therapy was not different from that observed with conventional chemotherapy in the ALL93 study. EFS and OS were estimated to be 60% and 76% at 1 year, and 49% and 57% at 2 year, respectively. Allogeneic SCT was performed for 49 patients (18 from a sibling donor, one from a related donor other than a sibling, 21 from an unrelated donor, and nine from unrelated cord blood), 39 (49%) of whom underwent SCT during the first CR with the median duration of 3.9 months (range, 1.2 to 18.0 months) from CR. Two patients received allogeneic SCT during the second CR. The probability for OS was 73% for those who underwent allogeneic SCT and 85% for those who did not (p=0.9416) at 1 year, and 49% and 57% at 2 year, respectively. Of 20 patients who experienced relapse, 17 were non-transplant cases at the time of relapse. Median CR duration for the 17 patients was 4.0 months (range, 2.8 to 12.4 months). Twenty-three patients died because of disease progression (n=6), complications during remission induction therapy (n=2), transplant-related causes (n=13), and external causes (n=2; melanoma and suicide). No significant effect of age, initial WBC counts, or type of BCR-ABL transcripts on survival duration was shown. Among 57 patients who achieved PCR negativity, 17 experienced recurrence of detection. Of them, 7 had a relapse, 6 proceeded to allogeneic SCT during first CR, and 4 remained disease-free without SCT.

The outcomes were compared with those for historical control patients from the JALSG ALL93 study [4] . Superiority of the present study was statistically significant for both EFS and OS (p<0.0001 for both). Separate analyses of patients who underwent allogeneic SCT during first CR showed that the probability of OS was almost identical (p=0.4820) in patients who underwent allogeneic HSCT with/without imatinib, but differed significantly for those who did not receive allogeneic HSCT (p=0.0006).

There is a general agreement that allogeneic SCT has been the only curative treatment modality for adults with Ph+ALL. The fact that a substantial proportion of patients do not qualify for allogeneic SCT due to the lack of a suitable donor, advanced age or underlying medical conditions, a non-transplant therapy with curative potential is eagerly looked-for. Imatinib has been the subject of hopeful anticipation because of its mechanism of action and of anti-leukemia activity demonstrated in the phase 1 and phase 2 single-agent studies [1, 2] . Although imatinib alone induced response in more than 50% of patients, but the response was not durable. This prompted us to plan a study to evaluate imatinibcombined regimen for newly diagnosed BCR-ABL-positive ALL. The result was excellent, achieving 96%CR and 71% PCR negativity, and confirming our preliminary observations [3] . It should be noted that other groups, such as the M. D. Anderson Cancer Center [5] and the German Multicenter ALL Study Group [6] ,19 also reported CR rates exceeding 90% and PCR negativity rates around 50% by imatinib-combined regimens.

For survival, imatinib-combined regimen appears to be superior to conventional chemotherapy. Although comparison with historical controls showed no significant differences in OS for those who underwent allogeneic SCT, patients treated with imatinib-combined regimen had a higher chance to receive allogeneic SCT. Patients who did not undergo allogeneic SCT, however, had significantly better survival than those treated with chemotherapy alone. When interpreting the results regarding survival, we should be cautious because of a relatively short observation period. Although longer follow-up is required to determine its effect on survival, our protocol clearly has a major potential to improve the treatment of Ph+ALL, one of the most therapeutically challenging hematologic diseases for which an effective treatment is yet to be developed. Invasive fungal infection is thought to be a major cause of morbidity and mortality in patients with leukaemia. Data from literature reviews suggests that among patients with invasive aspergillosis in about 43% the underlying disease is leukemia or lymphoma [1] . In these patients the case fatality rate is reported to be very high with 49%. However the true incidence and natural history of invasive fungal infections in patients with acute leukaemia is unknown. There are several reasons being responsible for this problem: 1. Diagnosis of invasive fungal infection has changed and improved very much during recent years. The advent of readily available high resolution pulmonary CT scanning has led to a large number of patients being identified as having probable invasive fungal infection [2] . In addition antigen assays and PCR for aspergillus species have improved our diagnostic sensitivity. Strict criteria for the definition of invasive fungal infections in the context of clinical trials have only recently been introduced [3] . 2. In clinical trials investigating treatment options in invasive fungal infections leukaemia is usually just one of several possible underlying diseases. For example in the voriconazole trial by Herbrecht et al only 43% of the patients had acute leukaemia [4] , whereas in the caspofungin trial by Walsh et al 73% of the patients had acute leukemia [5] . As it is not known what proportion of patients with acute leukaemia entered these trials, the true incidence and case fatality of invasive fungal infections in patients with leukaemia can not be calculated from these trials. 3 . In trials dealing with the treatment of leukaemia information on infectious complications often is incomplete. Thus mostly the total number of infections, and the treatment related mortality is reported, but no information is given on the specific diagnosis of the infection or on their fatality rate [6, 7] . Furthermore no reliable data are provided to show whether the diagnosis of invasive fungal infection meets accepted criteria. Thus it is difficult to precisely say how big the problem of invasive fungal infections in leukaemia really is. Although it is generally accepted that the problem is substantial, data on the true incidence and fatality rate of invasive fungal infections in leukaemia are incomplete. Abstract Much progress has been made in recent years in the treatment of patients with cancer. A new array of chemotherapeutic agents and biologicals has been introduced into treatment strategies leading to improved response and cure rates. The continuing expansion of stem cell transplantation by the use of reduced conditioning or haploidentical transplantation offers the chance of cure to many patients.

However the success of many of these treatment strategies is jeopardized by the occurrence of infectious complications leading to an increase in morbidity and mortality in these patients. The main reason for these infectious complications is the profound suppression of the innate and the acquired immune system. The single most important risk factor for infections is neutropenia. Degree and duration of neutropenia determine the rate and severity of infections. As patients with acute leukaemia are often treated with intensive chemotherapy they are therefore at especially high risk of developing severe infectious complications.

Optimizing anti-infective therapy has been in the focus of physicians treating patients with neutropenia for many years. Some standard principles of care have been clearly established: • Patients can be divided in at least two risk groups (low and standard). The most important denominator is the duration of neutropenia together with patient specific co-morbidities. Patients within the low risk group can be safely treated with oral antibiotics when they develop fever, while patients in the standard risk group have to be treated with i.v. antibiotics. • The most important sign of infection is fever. Patients who develop fever in neutropenia have to be treated immediately and appropriately. The concept of empirical treatment with antibiotics which cover the most important and most dangerous bacteria especially gram-negative organisms like pseudomonas aeruginosa has been widely accepted. Single drug therapy has been shown to be as good as a combination of two antibiotics in most patients.

• If fever persists, especially in patients with a long lasting neutropenia the possibility for an invasive fungal infection increases. Thus at least in patients in whom resolution of neutropenia is not imminent empirical antifungal drugs should be added to the antibacterial treatment.

Despite these standard principles of anti-infective treatment open questions still remain:

The prophylactic use of antibiotics in patients with neutropenia is a matter of debate. The widespread use of fluoroquinolones has been associated with the emergence of more gram-positive organisms causing infections over time. Especially the increased incidence of gram-positive bacteria like methicillinresistant Staphylococcus aureus (MRSA) and Streptococcus viridans strains may pose a major threat to immuno-suppressed patients [1] . While prophylaxis with fluoroquinolones in patients with neutropenia decreases the rate of febrile episodes and infection related events, the impact of widespread prophylaxis on the emergence of resistant organisms is uncertain. [2, 3] . Recent reports suggest that the emergence of multi drug resistant enterococci may be related to the widespread use of fluoroquinolones [4] . Thus the question in which patients in neutropenia the prophylactic use of antibiotics is useful, is still open. The definition of certain risk groups that might benefit or situations in which the benefit of prophylaxis outweighs the risk is necessary.

The addition of vancomycin to the initial treatment strategy of patients with neutropenic fever is still under debate. It has been shown in meta-analysis that successful treatment of infections in febrile neutropenic patients without modification of the initial antibacterial regime is more likely if a glycopeptide is added. This effect can be observed in patients with microbiologically documented infections as well as in patients with bacteremia and in patients with profound neutropenia. However no clear cut recommendation can be given as the overall mortality could not be reduced, the time to defervescence was not influenced and side effects are observed more often in the group of patients receiving additional glycopeptides [5, 6] . Whether the common practice to add glycopeptides if an infection with gram-positive bacteria or a central venous line infection is suspected or the patient suffers from severe mucositis is not definitely supported by evidence [6] .

Changing the initial antibiotic regime in persistently febrile patients Reassessment of patients after initiation of antibacterial chemotherapy has to take into account that the median time to response in patients with cancer treated for an infection may be as long as 5-7 days [7] . Thus reevaluation should probably not be done before day 3 to 5. If the patient continues to have fever and is still neutropenic several treatment options are suggested [8] : The patient may remain on the initial regime, it may be switched to another antibtiotic regime or the treatment with antibtiotics may be stopped and the patient be monitored closely thereafter. However evidence from clinical trials does not exist to definitely support one of these alternatives. Whether the switch to oral antibiotic drugs might be safe and useful, still needs to be evaluated as well.

When to switch antifungal therapy The addition of antifungals to patients with persistent fever in neutropenia is a valid option at least in patients at high risk of invasive fungal infection. In patients with proven or probable invasive fungal infection the use of an antifungal drug with efficacy against aspergillus species is recommended [9] . It is however not clearly defined when the initial antifungal treatment should be switched. The matter is complicated by the fact that radiologically an increase in pulmonary fungal lesions may occur with the resolution of neutropenia [10] , making it difficult to distinguish between progressive disease and response. Likewise it seems unclear for how long antifungal treatment has to be continued in patients with probable fungal infections, or which patients might benefit from secondary antifungal prophylaxis after resolution of neutropenia.

With an overall cure rate of approximately 80% achieved in contemporary clinical trials for childhood acute lymphoblastic leukemia (ALL), our ongoing Total Therapy Study XV at St. Jude Children's Research Hospital was designed not only to further increase cure rate but also to improve the patients' quality of life. The study consists of intensive systemic and intrathecal chemotherapy but does not include cranial irradiation, irrespective of a patient's risk features. The intensity of post-remission consolidation, continuation and reinduction therapy is based on the level of minimal residual disease at the end of the 6-week remission induction course. Thiopurine methyltransferase genotype and phenotype are determined prospectively for treatment modification. Methotrexate dosage is based on the risk of relapse and is further individualized to achieve optimal steady-state concentration. Preliminary results suggest that this regimen is likely to yield a cure rate approaching 90%. Molecular genetics, pharmacogenetic, pharmacodynamic, and gene expression profiling studies of host normal cells as well as leukemic cells, are performed in all patients and should further elucidate the mechanisms of drug resistance, leukemogenesis, and treatment-related toxicity.

While a cure rate of 80% can be achieved in some of the contemporary clinical trials for childhood acute lymphoblastic leukemia (ALL) [1] , refractory and relapsed forms of this disease still represent a leading cause of cancer-related deaths in children and pose formidable challenges. Moreover, a sizable proportion of the survivors of childhood ALL, especially those who had received cranial irradiation, will experience major and sometimes debilitating or fatal complications [2, 3] . Current efforts focus on optimization of the use of the existing antileukemic agents, early and systematic assessment of the risk of relapse, and pharmacogenetics of leukemic cells as well as host normal cells to avoid under-or over-treatment [2, 4] . In our ongoing Total Therapy study XV [5] , emphasis is placed not only on further improving cure rates but also on the reduction of long-term sequelae by limiting the cumulative doses of anthracyclines (100 mg/m2 in low-risk and 230 mg/m2 in standard-or highrisk cases) and cyclophosphamide (1 gm/m2 and 4.6 gm/m2, respectively), and by omitting cranial irradiation in all patients. Etoposide is given only to a small proportion of the patients (~5%) with high-risk leukemia who will subsequently undergo allogeneic hematopoietic stem cell transplantation.. Here we briefly describe the rationale of study design and the preliminary results.

Presenting age, leukocyte count, and leukemic cell immunophenotype and genotype are used for risk classification in virtually all contemporary clinical trials [6] . Measurement of minimal residual disease, by either flow cytometry or polymerase-chain-reaction analysis, is now recognized to be the most important prognostic factor because this measure reflects in vivo treatment response, encompassing both leukemic cell genetics and host pharmacogenetics; it also accounts for variations in drug absorption and interactions, and treatment compliance. This measure is not only more sensitive but also more specific than the conventional morphological examination [7] [8] [9] . Tandem application of both methods allow us to successful study minimal residual disease in virtually all patients [7, 9] .

In study XV, patients are assigned to three risk groups-low, standard-and high-risk (corresponding to standard-, intermediate or high, and very high-risk categories, respectively, by other study groups). B-cell precursor cases with age between 1 and 10 years and presenting leukocyte count <50x109/L, leukemic cell DNA index 1.16, or TEL-AML1 fusion are provisionally classified to have low-risk ALL, provided that they do not have testicular or central-nervoussystem (CNS) leukemia (i.e., CNS 3 status), hypodiploidy (<45 chromosomes), E2A-PBX1 fusion, or MLL rearrangement. Patients with BCR-ABL fusion (Philadelphia chromosome) are designated to have high-risk disease, and all others including all T-cell ALL cases are provisionally classified to have standard-risk ALL. Final risk status depends on the response to remissioninduction therapy. Any patients with 0.01% to 0.99% minimal residual leukemia after completion of 6-week induction therapy are considered to have standardrisk ALL and receive intensive postremission therapy. Patients with 1% or more residual disease are designated to have high-risk ALL and, together with Philadelphia chromosome-positive cases or those who require extended therapy to achieve morphologic remission, are candidates for allogeneic hematopoietic stem cell transplantation.

We also relate the pharmacogenetics of normal host cells and leukemic cells with treatment response and various treatment-related toxicities. In a previous study, we identified genetic polymorphisms of several drug-metabolizing enzymes and drug targets that influenced treatment outcome [10] . Whether different pharmacogenetic determinants will be identified in this study due to the use of substantially modified treatment remains to be determined. Importantly, our recent study showed that the acquisition of additional chromosomes in leukemic cells can create discordance between germline genotypes and leukemic cell phenotypes [11] . The finding emphasizes the benefit of studying pharmacogenetics of both leukemic cells and host normal cells.

We have shown that given the same dosage of high-dose methotrexate, T-cell leukemic blasts accumulate significantly less methotrexate polyglutamates than B-lineage blasts; among B-lineage cases, non-hyperdiploid blasts accumulate less polyglutamates than hyperdiploid blasts [12] . The findings support the use of increased dose of methotrexate (e.g., 5 gm/m2) in T-cell and higher-risk Bcell precursor cases [13] . More recently, we found that TEL-AML1-positive and E2A-PBX1-positive cases also accumulate less methotrexate polyglutamates than other B-lineage cases [14] . The use of high-dose methotrexate in our prior protocols may account for the excellent treatment results for cases with these genotypes that we treated in the 1990s [15] . The length of exposure is another important determinant of methotrexate polyglutamate formation and antileukemic effects in preclinical in vitro and in vivo models, with longer exposures at equal extracellular concentrations resulting in greater polyglutamylation and effects [16, 17] . Because findings from cell lines might not apply to patients, in this protocol we are conducting an upfront window study in which patients are stratified and randomized by initial leukocyte count (>versus<25x109/L), DNA index (versus<1. 16) , and leukemic cell immunophenotype (T-cell versus B-lineage) to receive methotrexate at a dose of 1 gm/m2 administered over 24 hours or the same dose over 4 hours. While both schedules should yield equivalent systemic exposure (i.e., the area under the plasma concentration-time curve), we will determine if methotrexate polyglutamate accumulation and pharmacodynamics differ between the two treatment groups and if the optimal duration of treatment differs between major ALL subtypes. Gene expression profile studies will also be performed before and after methotrexate treatment to extend our previous observations that leukemic cells of different molecular subtypes share a common pathway of genomic response to the same treatment, that the changes in gene expression are treatment-specific, and that drug cross-resistance patterns identify patients at very high risk of relapse [18, 19] . Additional studies may identify specific molecular target(s) or signaling pathway(s) for improved therapy.

Four days after methotrexate treatment (or immediately after diagnosis in patients who do not receive upfront methotrexate), remission induction therapy begins with daily prednisone (40 mg/m2 for 28 days), weekly vincristine (1.5 mg/ m2 for four doses), weekly daunorubicin (25 mg/m2 for two doses), and thrice weekly E coli asparaginase (10,000 units/m2 intramuscularly for 6 doses). Patients with 5% or more residual leukemia in bone marrow after 2 weeks of induction by morphologic or flow cytometry examination are given three additional doses of asparaginase. Subsequent induction therapy consists of cyclophosphamide (1,000 mg/m2) on day 26, mercaptopurine (60 mg/m2 per day) on days 26-39 and cytarabine (75 mg/m2) on days 27-30 and 34-37. Upon recovery of hematopoietic function, bone marrow is performed to determine remission status and the presence of minimal residual disease.

Consolidation/Reintensification therapy Consolidation therapy consists of high-dose methotrexate and age-adjusted triple intrathecal therapy with methotrexate, hydrocortisone and cytarabine (every other week for 4 doses) and daily mercaptopurine (50 mg/m2 per day) for 8 weeks. The dosage of methotrexate depends on the risk classification of patients, since higher dose (i.e., 5 gm/m2) is needed to improve outcome of T-cell and standard-/high-risk B-cell precursor ALL and lower dose (2.5 gm/m2) is adequate for low-risk B-cell precursor cases [13] . As we have shown that individualized doses based on the systemic exposure can improve outcome [20] , the dose is targeted to achieve a steady-state concentration of 65 M for standard-/high-risk cases and 33 M for low-risk cases.

Because high levels of minimal residual leukemia conferred a poor outcome even in the setting of allogeneic stem cell transplantation [21] [22] [23] [24] , reintensification therapy with high-dose cytarabine, etoposide, dexamethasone, and asparaginase is given to high-risk cases following consolidation therapy to maximize leukemic cell kill before allogeneic hematopoietic stem cell transplantation. In some cases, two courses of reintensification treatment are given to induce a molecular or immunologic remission (i.e., <0.01% leukemic cells in bone marrow). While transplantation with matched sibling donor is preferable, the procedure is also performed with matched-unrelated or haploidentical donor. To date, 16 patients have undergone hematopoietic stem cell transplantation for the following reasons: the presence of Philadelphia chromosome (n=4), initial induction failure (n=4) and minimal residual disease >1% at the end of 6-week remission induction.(n=8) Matched-related transplantation was performed in 7 patients, matched-unrelated transplantation in 5 and haploidentical transplantation in 4.

In the first 20 weeks of continuation therapy, low-risk cases receive daily mercaptopurine (75 mg/m2) and weekly methotrexate (40 mg/m2) with pulses of daily mercaptopurine (75mg/m2), dexamethasone (8 mg/m2 per day in three divided doses for 5 days) and vincristine (2 mg/m2) given every 4 weeks; standard-risk cases receive daily mercaptopurine (50 mg/m2), weekly native E coli asparaginase (25,000 units/m2), and doxorubicin (30 mg/m2) plus vincristine (1.5 mg/m2) every three weeks. All patients receive reinduction therapy twice (weeks 7-9 and weeks [17] [18] [19] [20] during the first 20 weeks of continuation therapy. Reinduction therapy in low-risk cases consists of dexamethasone (8 mg/m2 on days 1-8 and days [15] [16] [17] [18] [19] [20] [21] , vincristine (1.5 mg/m2 weekly for 3 doses), asparaginase (10,000 units/m2 thrice weekly for 9 doses) and doxorubicin (30 mg/m2 on day 1). In standard-risk cases, reinduction therapy consists of dexamethasone and vincristine (doses same as those in low-risk cases) as well as asparaginase (25,000 units/m2 on days 1, 8 and 15), plus doxorubicin (30 mg/ m2 on days 1 and 8) in the first course, or high-dose cytarabine (2 gm/m2 every 12 hours for four doses on days 15 and 16) in the second course. In this regard, double reinduction therapy has been shown to improve outcome of standard (or intermediate)-risk and high-risk ALL cases with poor early response [25, 26] . Preliminary result suggested that the interrupted use of dexamethasone reduced the risk and severity of osteonecrosis [13] but it is not certain if this schedule might compromise the antileukemic efficacy of this agent.

The remaining continuation therapy in low-risk cases consists of daily mercaptopurine (75 mg/m2) and weekly methotrexate (40 mg/m2), interrupted by pulse therapy every 4 weeks (up to week 100) with dexamethasone (8 mg/m2 per day in 3 divided doses for 5 days), vincristine (2 mg/m2) and mercaptopurine (75 mg/m2 per day for 7 days). In standard-risk cases, the remaining continuation therapy consists of 3 drug pairs given in 4-week blocks: mercaptopurine (75 mg/m2 daily for 7 days) plus methotrexate (40 mg/m2 per week) in the first and second weeks, cyclophosphamide (300 mg/m2) plus cytarabine (300 mg/m2) in the third week (to be replaced by mercaptopurine and methotrexate after week 67), and dexamethasone (12 mg/m2 per day in 3 divided doses for 5 days) plus vincristine (2 mg/m2) in the fourth week (to be replaced by mercaptopurine and methotrexate after week 100).

The dosages of mercaptopurine and methotrexate are tailored to the limits of tolerance (as indicated by the total leukocyte and absolute neutrophil counts) but caution is taken to avoid overzealous escalation of dosages leading to frequent interruption of chemotherapy and overall low-dose intensity, which has been associated with an inferior outcome [27] . Methotrexate is given intravenously to ensure compliance [15] . Thiopurine methyltransferase phenotype and genotypes are determined prospectively in all patients. The dosage of mercaptopurine is reduced in those with low enzyme activity to avoid excessive hematopoietic toxicities and to decrease the risk of therapy-related cancers [28, 29] .

Although cranial irradiation is the most effective CNS-directed therapy, its use has been limited to patients at high risk of CNS relapse in contemporary clinical trials because of the associated neurocognitive sequelae, endocrinopathies, and second tumors [2, 3] . We have also shown that prior treatment with cranial irradiation was associated with low employment rate, low marriage rate (in women), and an excess of late mortality due to second cancer in long-term survivors of ALL [3] . In fact, the cumulative risk of second tumor exceeded 20% at 30 years after initial remission. In Study XV, we will determine if cranial irradiation can be omitted in all patients and replaced by risk-directed systemic and intrathecal therapy. Cranial irradiation is reserved only for patients who develop a CNS relapse. Because traumatic lumbar puncture at diagnosis increased the risk of CNS relapse [30, 31] , we routinely perform this procedure under deep sedation or general anesthesia, transfuse patients with thrombocytopenia (i.e., <100 x 109/L), and instill age-adjusted intrathecal cytarabine immediately after collection of cerebrospinal fluid [32] . Moreover, the procedure is performed only by the most experienced clinicians because experience is one of the most important determinants of a successful lumbar puncture [33] .

Age-adjusted triple intrathecal chemotherapy with methotrexate, hydrocortisone, and cytarabine is given on day 19 and at the end of remission induction (coinciding with the start of consolidation therapy with high-dose methotrexate). Because of their increased risk of CNS relapse, additional intrathecal therapy is given on days 8 and 26 of remission induction in patients with CNS 2, CNS 3, traumatic lumbar puncture with blasts, T-cell ALL with leukocyte count >50x109/L, B-cell precursor ALL with leukocyte count >100x109/L, or the presence of Philadelphia chromosome, MLL rearrangement, or hypodiploidy <45 chromosomes [32] . Triple intrathecal chemotherapy is given every other week during consolidation therapy, and then every 8 weeks in low-risk cases and every 4 weeks in standard-risk cases up to one year (or every 4 weeks to the time of transplantation in high-risk cases); those at particularly high risk of CNS relapse (i.e., T-cell ALL with leukocyte count >50x109/L, B-cell precursor ALL with leukocyte count >100x109/L, presence of Philadelphia chromosome, MLL rearrangement or hypodiploidy<45, or CNS 3 status) continue to receive intrathecal therapy every 8 weeks beyond the first year until week 96 of continuation therapy.

Of the 274 patients aged 1 to 18 years enrolled onto the study from July 2000 to August 2005, 268 (97.8%) attained complete remission after 6 weeks of remission induction. Of the 6 initial induction failures, 2 were due to fatal bacterial infection and the other 4 were due to refractory leukemia. All 4 refractory cases eventually achieved remission with extended induction therapy. Postremission failures included hematological relapse in 5 cases, CNS relapse in 3, and lineage switch, secondary myelodysplasia, and death in remission in one case each. Notably, all three CNS relapses occurred within the first year of continuation treatment; because prophylactic cranial irradiation was scheduled at one year of continuous complete remission in our clinical trials in the 1990s, their CNS relapse would not have been prevented by irradiation even if they were treated in our prior studies. The overall treatment result is promising,: 4year event-free survival and overall survival rates (SE) are 92% (4.0%) and 96% (3%), respectively [34] . This improved result has eliminated the impact of many prognostic factors once associated with poor outcome, e.g., male sex and high initial leukocyte count. Besides the presence of Philadelphia chromosome, the only adverse prognostic factors consisted of positive minimal-residual-leukemia (>0.01%) at the end of 6-week remission induction for event-free survival, and age > 10 years at diagnosis for survival. Key words: Acute lymphoblastic leukemia, Cranial irradiation, Central-nervoussystem leukemia With the achievement of 5-year event-free survival rates of 80% or more in some contemporary clinical trials for childhood acute lymphoblastic leukemia (ALL), recent effort has focused on optimal risk-directed therapy to avoid overor under-treatment. Because cranial irradiation can cause many acute and late complications, including second cancers, late neurocognitive deficits, and multiple endocrinopathies [1] , this treatment modality has largely been replaced by intensive intrathecal and systemic chemotherapy, and radiation is now reserved only for patients at high risk of central-nervous-system (CNS) relapse. It has been well recognized that high-risk genetic features, T-cell immunophenotype, a large leukemic cell burden, and CNS leukemia are associated with an increased risk of CNS relapse. In 1993, we reported that the presence of any number of identifiable leukemic cells in cerebrospinal fluid at diagnosis also conferred an increased risk of CNS relapse, and proposed a new classification of CNS status at diagnosis: CNS1 denotes the absence of identifiable leukemic blast cells in cerebrospinal fluid; CNS2 the presence of leukemic cells in a sample that contains fewer than 5 white blood cells/L; and CNS3 a nontraumatic sample that contains 5 or more white blood cells/L with identifiable blasts in cerebrospinal fluid, or the presence of a cerebral mass or cranial nerve palsy [2] . Although the prognostic impact of CNS2 status on eventfree survival has since been shown to be treatment-dependent, patients with this feature still have 2-to 3-fold increased risk of CNS relapse in many clinical trials [3] [4] [5] [6] . In a subsequent study, we reported that traumatic lumbar puncture with identifiable blast cells in cerebrospinal fluid at diagnosis of ALL was also associated with an increased risk of CNS relapse and a poor event-free survival [7] , a finding confirmed by two recent studies [6, 8] . The adverse consequence of traumatic lumbar puncture with blasts may not only be due to the iatrogenic introduction of blast cells into CNS but could also be due to less effective subsequent intrathecal therapy because of poor distribution of drugs in the CNS due to a hematoma in the epidural or subarachnoid space, or to scarring or segmentation of the subarachnoid membrane. Recognizing the adverse impact of traumatic lumbar puncture with blasts, we intensified triple intrathecal therapy for these patients in Total Therapy study XIIIB, as had been done for patients with a CNS2 status [9] . In this study, cranial irradiation was administered to 12% of the patients with either T-cell ALL and a leukocyte count of 100x109/L or with CNS3 status. This approach resulted in a 5-year cumulative incidence of isolated CNS relapse of 1.7%±0.8%, and of any CNS relapse of 3.0%±1.1%; none of the patients with traumatic lumbar puncture with blasts developed CNS relapse in this study. Use of dexamethasone during the two reinduction treatments and throughout continuation therapy, may also have contributed to the improved CNS control in this study.

Although intensive intrathecal therapy can abolish the poor prognostic impact of a traumatic lumbar puncture with blasts, this treatment can also adversely affect neurocognitive and spinal cord function [10] [11] [12] . Hence, every effort should be made to avoid traumatic lumbar puncture. In our current Total Therapy study XV, we routinely perform lumbar puncture with patients under deep sedation or general anesthesia. Only the most experienced clinicians perform the diagnostic lumbar puncture and thrombocytopenic patients are transfused with platelets before the procedure because inexperience and thrombocytopenia are important risk factors for traumatic lumbar puncture [13] . We also administer intrathecal cytarabine immediately after the collection of diagnostic cerebrospinal fluid. Previous practice of performing a diagnostic lumbar puncture followed in one or two days by another procedure to administer intrathecal therapy might have adversely affected the distribution of drugs, owing to contraction or collapse of the subarachnoid space due to leakage of the cerebrospinal fluid or to external compression by extra-axial blood or spinal fluid, preventing access to free-flowing cerebrospinal fluid. Other measures to prevent CNS relapse in Total XV study include the use of high-dose methotrexate targeted to achieve optimal systemic exposure; dexamethasone during reinduction and continuation therapy; and early intensification of triple intrathecal therapy [14] . Cranial irradiation is not given to any patients regardless of risk features, and is reserved only for those who develop CNS relapse.

Of the first 274 patients enrolled onto Total therapy study XV from July 2000 to August 2005, the rate of traumatic lumbar puncture was 7.5% and that of traumatic lumbar puncture with blasts was 4.8%, which are substantially lower than the historical rates of 29% and 11%, respectively [7, 13] The 4-year eventfree survival rate (SE) is 92% (4.0%) [15] . Isolated CNS relapse occurred in three patients, resulting in a 4-year cumulative rate of 1.9% (0.9%). One patient with CNS relapse had B-lineage ALL and CNS2 status at diagnosis, whereas the other two had T-cell ALL and leukocyte count >100x109/L with CNS2 status and traumatic lumbar puncture with blasts at diagnosis, respectively. Hence, despite intensive triple intrathecal and systemic therapy, patients with T-cell ALL and leukocyte count >100x109/L have a relatively high rate of CNS relapse with a 4-year cumulative rate of 16.3% (11.7%). It may be of interest to test if the CNS relapse hazard in these patients can be reduced by modestly altering CNSdirected therapy, for example, by the use of intrathecal liposomal cytarabine. As overall treatment efficacy improves, it becomes increasingly important to optimize various components of therapy, so as to minimize toxicity while maintaining or enhancing efficacy.

Intensive research has been carried out to identify the processes and mechanisms of leukemogenesis. In the following article, we would like to give a short overview of childhood acute myeloid leukemias (AML).

Further, the evolution from predisposition to overt leukemia will be illustrated by some specific models of childhood AML.

Whereas some steps of leukemogenesis are well defined, several mechanisms remain hypothetical although supported by experimental studies in humans, mice or cell lines.

AML are clonal disorders of stem cells, common myeloid-lymphoid or early myeloid progenitors. Bonnet and Dick demonstrated the hierarchical differentiation of AML resembling in part normal hematopoiesis [2] . Like normal hematopoietic stem cells (HSC) the leukemic stem cell (LSC) is characterized by the capacity of self-renewal, proliferation, remnant differentiation programs and maturation [12] . Different stages of leukemic blast differentiation (preLSC, LSC, leukemic progenitor [LPC], leukemic blast) in associated with specific features could be defined (table 1) .

Gilliland et al. [9] showed that in most leukemias different types of mutations are necessary to develop overt leukemia. Whereas class II mutations such as t (8;21), t(15;17) or inv(16) mainly cause impaired differentiation and maturation, additional class I mutations involving regulatory receptors (FLT-3, c-kit) or cell cycling associated proteins (ras) lead to increased proliferation [27] .

The preleukemic stem cell (preLSC) defined as clone with class II mutation but no clinical symptoms or evidence for blasts in morphology, is characterized by self-renewal, an almost normal duration of cell cycle, the capacity of differentiation and proliferation. It could be supposed that they have a defined and thereby limited life span. The clones with translocations t(8;21), t(12;21) or inv(16) persist without causing clinical symptoms prior frank leukemia or after remission up to several years [12] .

So far, two models of LSC origin could be considered as possible. 1) The LSC derives from a multilineage progenitor maintaining self-renewal capacity. 2) The LSC derives from a mutated lineage restricted precursor regaining the selfrenewal [4] . Several studies give evidence that the initial leukemic event occurs in differently differentiated stem cell compartments [6] , associated with prognosis.

The evolution from a preLSC to a LSC is supposed to be associated with the occurrence of class I mutations such as FLT-3 internal tandem duplications (ITD) or c-kit mutations. FLT-3-ITD leads to the loss of external, FLT-ligand mediated regulation. The dimerization of the membrane receptors causes an auto activation of proliferation. In childhood AML the frequency of FLT-ITD was found to be 12%. Moreover, in core-binding factor leukemias (CBF-AML) the frequency was even higher (17%) [27] and associated with an impaired prognosis. Also c-kit (11.5%) and and ras mutations (18%) could be identified in a relevant percentage of children [11] . Less frequently PTPN11 mutations [10] , typically detected in monoblastic leukemias, have been identified in our patients group.

The different types of mutations do not necessarily occur at the same time point (figure 1).

The studies by Greaves and others identified a high frequency of recurrent leukemia associated mutations in spots of Guthrie screening cards from newborns supporting a prenatal origin of the first leukemogenic event [12] . However, only less than 1% of the perinatally detected preleukemic clone developed frank leukemia later on. The latency until leukemia was up to 11 years [12] . The low frequency of leukemias compared to the number of detected mutations suggests physiological mechanisms which are able to eliminate the preleukemic stem cell. Possible explanations comprise a limited life span of the LSC or changes in the environment not longer supporting the (pre-) leukemic clone. This is also supported by the long lasting persistence of leukemia associated fusion genes after successful treatment of the AML.

So far, neither the intracellular events and mechanism of regulation by transcription factors nor the interaction between leukemic precursors and stroma cells are completely defined.

Whereas most AML in children do not differ from those of adults in terms of cytogenetics, immunophenotypes or morphology a few entities are almost unique in children. These are monoblastic leukemias in infants associated to MLL-rearrangements, megakaryoblastic leukemias with t(1;22), acute megakaryoblastic leukemias in children with Down syndrome (DS-AMKL) and AML associated with Fanconi-anemia or neutropenias such as Kostmann-or Especially, the DS-AMKL might be an excellent model for leukemogenesis. Children with Down syndrome are at a 150 fold risk to suffer AMKL. The genetic background is defined by the trisomy 21 and the recently detected mutations of the hematopoietic TF GATA1 in nearly all patients. In addition, this type of leukemia occurs within a narrow range of age (1st to 4th year of age). Further, the transient myeloproliferative disorder (TMD) in newborns with trisomy 21, which is characterized by a leukemia-identical morphologic and immunological phenotype but surprisingly by a spontaneous remission, could help to identify leukemogenetic features (figure 2) [13] . Finally, the AMKL in children without Down syndrome exhibit similarities in terms of blast phenotype and age at diagnosis but a very high resistance to treatment, providing excellent options for comparison to DS-AMKL. Therefore here we exemplary described the evolution of leukemogenesis in children with DS.

As proposed by others, the trisomy 21 could be assumed as both, predisposition and first event (class II mutation) in leukemogenesis. Studies in murine embryonic hematopoiesis demonstrated an increased and prolonged turnover of myelo-and megakaryopoiesis. This is supported by the finding that children with trisomy 21 have increased levels of platelets compared to non-trisomy 21 patients. As RUNX1, ETS-2, ERG and BACH1, hematopoietic transcription factors involved in early hemato-and megakryopoiesis are encoded on chromosome 21, a gene dosage effect might be the most likely origin. However, a general gene dosage effect could not be confirmed. A recent study showed that an increase of gene expression is tissue specific and developmental-stage dependent [23] . Analysis of bone marrow progenitors from children with trisomy 21 did not show increased expression of RUNX1, ETS-2 or ERG. On the other hand, in regenerating bone marrow from children in remission after DS-AMKL, a significantly increased expression of RUNX1 and ETS-2 could be shown accompanied by an increased amount of RUNX1 depended antigen expression such as CD56 and CD36 [20] . Although the definitive confirmation in stimulated fetal hematopoiesis is lacking, a RUNX1 over expression during stress hematopoiesis might explain the megakaryopoietic turnover.

Regarding trisomy 21 as a predisposition for leukemias, it does not explains why the AMKL only affects children less than 5 years of age. A specific susceptibility can be supposed. Li et al. demonstrated the intrauterine restricted effects of the mutated GATA1s in mice. This arises the possibility that the target cells in other leukemias of infancy and early childhood are distinct from those in adult leukemias [16] .

For the evolution of a leukemic cell, an environment which provides a cell survival and proliferation advantage of the aberrant clone is required. In the trisomy 21 model several studies suggest that an increased megakaryocytic secretion of transforming growth factor (TGF) [3] or platelet derived growth factor (PDGF) induces an environment with advantages for an increased myeloand megakarypoiesis [1, 19] . This thesis might be supported by increased levels of thrombopoietin (TPO) [24] , which is involved in both, early myeloproliferation and megakaryopoiesis.

These conditions might cause a positive selection of the preleukemic GATAs-clone and favor additional events.

In children with Down syndrome the mutation in exon 2 of the GATA1 causes the loss of the N-terminal activation domain leading to a shorter gene product (GATA1s) with decreased activity [14, 26] . The data provided by Li et al. show that in murine fetal hematopoiesis GATA1s exerts dominant action on early progenitors leading to hyperproliferation [16] . Analyzing the expression of hematopoietic TFs we could demonstrate a significant increase of GATA1s expression in blasts derived from children with TMD or DS-AMKL ( figure 2) . Similarly, the early acting GATA2 was increased. By contrast, the chromosome 21 encoded TFs RUNX1, ETS-2 and ERG were not differently expressed compared to healthy controls with and withour Down syndrome.

Due to the loss of the N-terminal site in GATA1s, the binding site of RUNX1 [8] , we suppose that an impaired ability to form a transcription complex with RUNX1, causes an inhibition of megakaryocytic differentiation and maturation but not proliferation.

Further, the balance between GATA1 and GATA2 is disturbed [18] and due to the reduced activity of GATA1s GATA2 is up regulated (figure 3). Increased expression of GATA2 has been shown to stimulate megakaryo-and myelopoietic proliferation but to inhibit maturation.

Another finding is the reduced expression of PU.1 in TMD and DS-AMKL blasts ( figure 3 ).

In addition, some studies provide evidence that GATA1 interferes directly with PU.1 [5] . Decrease to critical expression levels but not the complete loss of expression of PU.1 has been shown to be involved in the development of AML [21] .

The development of an overt leukemia requires not only the clonal expansion of the degenerated blast but also a supporting environment. The malignant clone itself can contribute to establish conditions for his growth advantage.

In the example of the DS-AMKL, this might be the continuation of elevated TGF secretion from the malignant and normal megakaryoblastic precursors. As TGF typically inhibits tumor and leukemic growth, specific escape mechanisms of the megakaryoblastic clone are necessary to achieve an absolute and relative dominance. Whereas the stroma cells either in the fetal liver (TMD) or in bone marrow (DS-AMKL) experience induction of fibrotic changes (as displayed in liver -or myelofibrosis), the megakaryoblasts seem not to be inhibited by TGF. Indeed, the examination of TGF-receptors (TBR) showed a marked downregulation of the TBR III (figure 4), which might prevent the effects of TGF.

Similar mechanism of tumor cell escape have been demonstrated in renal carcinomas, depending on stage [7] . In addition the TGF pseudoreceptor BMP and membrane bound inhibitor (BAMBI) were strongly up regulated (figure 4). BAMBI binds TGF, but due to the lack of the intracellular tyrosine kinase domain, the TGF signaling was inhibited. In addition, BAMBI interferes with TBR I, leading to a further inhibition of TGF effects [17] . These mechanisms have already been shown in colorectal tumors and hepatocellular carcinomas [22] .

The leukemic blasts represent the dominant phenotype of the leukemias. Typically rudimental features of normal differentiated cell are expressed. Analysis by immunophenotyping reveals in most cases blasts with differently expressed antigen patterns reflecting hematopoietic development ( figure 5 ). Comparison of the immunophenotype at diagnosis and relapse supports the model of the LSC because the percentage of blasts expressing stem antigens at relapse increases significantly [15] . Monitoring of the blast cell population during treatment shows a fast elimination of the more differentiated blasts, defined by lineage specific antigens, but a somewhat slower decline of blasts expressing stem cell associated antigen patterns.

Therapy of childhood AML requires an intensive cytostatic, cytarabine and anthracycline based, treatment. However, cytostatic drugs preferentially affect dividing cells. Given that the preLSC or LSC maintain or regain the ability of quiescence additional mechanisms of elimination are needed. Studies monitoring residual disease show that in several cases a leukemic clone remains detectable [25] . Interestingly, this does not necessarily predict relapse. Whereas immunological effects to treat residual disease are common after allogenic stem cell transplantation (graft versus leukemia effect), an immunotherapy against LSC seems to be difficult. Therefore, changes in the environment or a limited life span of the LSC are more likely the reason of final preLSC elimination.

In conclusion, a better knowledge of the LSC characteristics and the environment will help to identify new ways to treat childhood leukemia or even to treat the preLSC. Keywords: Childhood acute myeloid leukemia, Treatment, Outcome.

Substantial progress in the management of childhood acute myeloid leukemia (AML) has been observed over the past 20 years [8] . However, about 40% of patients with AML experience relapse, and many survivors face serious longterm effects of treatment with large doses of anthracyclines, alkylating agents, blood products, antibiotics, and/or radiation therapy. Since the mid-1970s, St. Jude Children's Research Hospital (St. Jude) has designed and conducted clinical trials for children and adolescents with AML. This presentation will discuss the efficacy and toxicity of the consecutive St. Jude AML studies and will suggest areas for further improvement. The long-term outcomes of patients treated on four consecutive front-line St. Jude AML studies (AML-805, AML-836, AML-871, and AML-919) have recently been updated [10] . The AML-80 protocol (enrollment, 1980 through 1983)5 compared allogeneic hematopoietic stem cell transplantation (HSCT) with sequential intensive chemotherapy for patients in first complete remission (CR). The overall CR rate was 80%. The 15-year event-free survival (EFS) estimate was 44.4%±14.8% (SE) for the 9 patients who underwent HSCT and 26.8%± 5.7% for the 56 patients treated with chemotherapy alone. However, after adjusting for time to transplant, we observed no statistically significant difference in overall (p=0.52) or event-free (p=0.76) survival between these groups. The AML-83 study (1983) (1984) (1985) (1986) (1987) [6] was based on the hypothesis of Goldie and Coldman that maximal early eradication of leukemia cells by using multiple noncross resistant chemotherapy agents would improve outcome. Patients in CR received 16 cycles of post-remission non-ablative chemotherapy. HSCT was not offered to patients in first CR. Although the remission rate was 88.9%, the 15year EFS estimate of these patients was only 8.9%±3.8%. In the AML-87 protocol1 (1987) (1988) (1989) (1990) (1991) , the dosage of etoposide and Ara-C was adjusted on the basis of plasma drug concentration to optimize systemic exposure. The overall rate of CR was 82%. About 3.5% of patients died from complications of early therapy. Reflecting the intensity of the chemotherapy regimen, the death rate during remission in the postremission therapy phase was relatively high, (about 10%). The 10-year EFS and overall survival estimates were 33.3%±7.3% and 48.7%±7.8%, respectively. Long-term survivors have experienced several chronic medical problems. In the AML-91 protocol9 (1991 through 1996), all patients began therapy with cladribine (2-CDA) alone; patients who had a complete or partial response received another course of 2-CDA before proceeding to postremission chemotherapy and either autologous or allogeneic HSCT during first remission. Retroviral gene-marking was used to determine the origin of relapse after autologous transplantation [2] . Patients whose leukemia did not respond to 2-CDA received two or three courses of daunomycin, Ara-C, and etoposide (DAV) before proceeding to transplant. The overall remission rate was 79%. The EFS and overall survival estimates were 43.5%±6.3% and 56.5%±6.3% at 5 years and 41.8%±10.6% and 53.0%±7.0% at 10 years. After adjustment for time-to transplant, patients receiving chemotherapy alone were not at significantly greater risk of an adverse event than patients receiving autologous transplants (p=0.43) or allogeneic transplants (p=0.39).

The AML-97 protocol4 (1997-2002) built on the AML-91 study. It consisted of 2-CDA given by 2-hour bolus infusion plus Ara-C administered either as a 2hour bolus infusion (Arm A) or a continuous infusion (Arm B). The combination of Ara-C with 2-CDA was chosen because of evidence that giving Ara-C with another nucleoside analogue increased the accumulation of ara-CTP in the circulating blast cells in both adult and pediatric AML. Initially, all patients received postremission HSCT. Patients with high-risk AML [monosomy or deletion 7, 5q, trisomy 11 or 8, t(9;22), secondary AML, megakaryoblastic leukemia, or absence of remission after first course of DAV] were candidates for allogeneic HSCT. Others were eligible for autologous HSCT; autologous marrow was purged by two different methods and marked with a retroviral vector. In 1998, because the retroviral marker was not available, the protocol was amended to substitute two courses of intensive chemotherapy for autologous HSCT. Between March 1997 and May 2002, 78 children with newly diagnosed, untreated primary AML were enrolled on the St. Jude AML-97 protocol. Thirty-eight patients were randomly assigned to receive 2-CDA plus short daily infusions of Ara-C, and 40 were assigned to receive 2-CDA plus continuous infusion of Ara-C. Forty-nine patients' bone marrow samples contained sufficient leukemic blast cells for pharmacokinetic and pharmacodynamic studies (21 patients in Arm A and 28 in Arm B). The leukemia cells of patients on Arm A (receiving short daily infusions of Ara-C) contained significantly higher median ara-CTP levels than those of patients on Arm B (receiving continuous-infusion Ara-C) on both day 1 (p=0.0378) and day 2 (p=0.0220). No significant difference was observed between Arm A and Arm B in inhibition of DNA synthesis on day 1 or day 2 of treatment. Of 78 patients enrolled in the AML-97 study, one died of fungal infection during ara-C and 2-CDA therapy and was not evaluable for response. Of the 77 evaluable patients, 43 (56%) achieved CR after one course of ara-C and 2-CDA, 69 (90%) achieved CR after a course of DAV, and 74 (96%) achieved CR after an additional course of DAV induction therapy. A CR was documented in 16 of 37 patients (43%) after short-infusion Ara-C plus 2-CDA (Arm A) and in 27 of 34 patients (67%) after continuous-infusion Ara-C plus 2-CDA (Arm B; p=0.0404). After the first course of DAV, 30 of 37 (81%) Arm A patients and 39 of 40 (98%) Arm B patients achieved remission (p=0.025). After the second course of DAV, all 40 patients in Arm B and 34 of 37 (92%) in Arm A had achieved remission (p= 0.106). The estimated 3-year EFS for the entire cohort is 48.1%±6.1%. The EFS did not differ significantly by treatment arm (p=0.467) or by karyotype (p=0.106). The estimated 3-year overall survival for the entire cohort is 54.5% ±6.7%. Overall survival did not differ significantly by arm (p = 0.577) or by karyotype (p=0.385) in the study group as a whole. The AML-97 study also assessed the usefulness of a 4-color flow cytometric strategy developed in our laboratory to study residual disease. About 85% of the newly diagnosed cases of AML had immunophenotypes that allowed detection of 0.1%-0.01% residual leukemic cells. The 2-year survival estimate was 33.1%±19.1% for patients with 0.1% AML cells by flow cytometry after induction therapy but 72.1%±11.5% for those with <0.1% AML cells (P=0.022); overt recurrence of AML within the subsequent 6 months was significantly more likely in the former group [3] . The current St. Jude AML study, AML-0212, assigns therapy according to risk and leukemia genotype. Risk factor stratification is similar to that of the AML-97 study but incorporates FLT-3 internal tandem duplication (ITD) status and information about the amount of minimal residual disease (MRD) after each cycle of induction therapy. Patients with FLT-3 ITD are considered at high risk of relapse regardless of other features. For patients with MRD 1% after the first cycle of induction chemotherapy, Mylotarg is added either alone or in combination with Ara-C, daunomycin, and etoposide. Allogeneic HSCT (with a matched related or unrelated donor) is recommended during first CR for patients at high risk of relapse and for patients at standard risk of relapse if a related matched donor is available. Because outcome was not influenced by the Ara-C schedule or intracellular incorporation of Ara-CTP in AML-97, patients are randomly assigned to receive Ara-C on a high-or standard-dose schedule. Preliminary results of the first 112 patients enrolled on AML-02 were recently presented [12] . The overall rate of CR has been excellent regardless of the initial randomization. Only three patients had not obtained a CR (resistant AML, 2 patients; infectious complication, 1 patient) after the induction II phase (CR rate, 97%). The 1-year event-free and overall survival estimates are 76.8%±5.1% and 89.1%±3.8%, respectively. As a group, AML cases with FLT-3 ITD have fared poorly: only 1 of 11 patients was MRD-negative after Induction I and only 3 were MRD-negative after Induction II. Initially, central nervous system (CNS)directed therapy consisted of intrathecal Ara-C alone. Because CNS relapse occurred in 3 of the first 33 patients treated, intrathecal therapy was changed from Ara-C alone to triple combination therapy with methotrexate, hydrocortisone, and cytarabine; none of the 79 subsequent patients has had a CNS relapse.

The St. Jude consecutive clinical trails have provided important lessons in the management of children and adolescents with AML and a framework for future studies. As noted in AML-80, AML-91, and many other studies, the role of HSCT in the overall management of AML remains to be established. AML-83, which had the poorest results of the St. Jude AML trials, revealed that when remission is achieved with non-myeloablative cycles of chemotherapy, the longterm results are dismal even with prolonged maintenance therapy. Despite its overall poor results, AML-83 demonstrated that subtypes of AML defined by morphology or genetic features respond differently to treatment. The rate of CR after etoposide plus Ara-C was almost twice as high in the morphologically defined FAB M4 or M5 subtypes as that in other FAB subtypes. Further, aggregate data from AML-80 and AML-83 showed that the 2-year EFS estimate of patients with M5 AML and the t(9;11) was 75% [11] . In the AML-87 protocol, one of the most intensive St. Jude AML regimens to date, the dosages of both cytarabine and etoposide were adjusted to achieve a targeted plasma concentration. As expected, the combination delivered on this schedule caused severe gastrointestinal toxicity, which usually delayed the next course of chemotherapy. Despite the intensity of treatment (a 15% death rate during remission from infectious complications) and the use of HSCT for selected patients in second remission, the 10-year survival rate was only 40%. This and other studies of pediatric AML demonstrate that the indiscriminate intensification of early therapy may reduce overall survival rates by increasing mortality caused by toxicity or by unduly delaying subsequent consolidation therapy. In AML-91, which used single-agent 2-CDA to induce remission, the remission rate of 78% was comparable to those of previous St. Jude AML trials. As in AML-83, patients with M5 AML, the t(9;11), or both had very good responses to 2-CDA7. However, patients with acute megakaryoblastic leukemia had very poor responses (no CR among 10 patients). In the AML-97 study, which randomly assigned patients to two different schedules of Ara-C in combination with 2-CDA in the first induction cycle, the CR rate was significantly higher with continuous infusion of Ara-C but was not correlated with intracellular accumulation of Ara-CTP or inhibition of DNA synthesis. In that protocol, we began to systematically assess the clinical significance of MRD. In the current AML-02 protocol, information about MRD is being used to guide treatment choices. However, contrary to findings in acute lymphoblastic leukemia, the absence of residual AML after completion of induction therapy does not virtually assure that relapse will not occur.

In summary, the St. Jude experience suggests that early intensive chemotherapy with excellent supportive care produces remission rates greater than 90% in children and adolescents with AML. However, our results and those of others show that about 40% of cases of AML are highly resistant to current therapy and that further intensification of therapy is unlikely to improve outcome. CNS-directed therapy has also been a point of contention among study groups and has not been systematically studied. The St. Jude experience suggests that risk-directed triple intrathecal chemotherapy in the context of intensive systemic chemotherapy is likely to be effective even for patients with CNS leukemia, and that cranial irradiation may not be necessary. Future clinical trials for AML should identify specific treatment questions for specific subsets of patients. For example, AML with FLT-3 ITD appears to be resistant to conventional chemotherapy and perhaps to HSCT. It is possible that the use of FLT-3 inhibitors in front-line therapy might improve the outcome of this subgroup of patients. Patients with acute megakaryoblastic leukemia, biphenotypic leukemia, or AML associated with myelodysplastic features, who are unlikely to benefit from intensive chemotherapy, could be invited to participate in innovative trials. Finally, because of the small number of patients in specific subgroups of pediatric AML, clinical investigations should be conducted as multi-institutional collaborations. Through the combination of all-trans retinoic acid (ATRA) and chemotherapy, cure is now a reality for most patients with acute promyelocytic leukemia (APL). In fact, several modern approaches based on this combination have led to prolonged disease-free survival and potential cure for more than 80% of patients achieving complete remission. The current consensus on the most appropriate induction therapy, once a diagnosis of APL has been confirmed at the genetic level, consists of the simultaneous administration of ATRA and anthracyclinebased chemotherapy. The choice of anthracycline and whether it should be combined with other agents, such as cytosine arabinoside, remain controversial. Exceptions to the use of anthracycline-based induction regimens should be considered only for individual patients in whom chemotherapy is contraindicated. This is the case of patients with certain clinical conditions such as severe organ failure, anticoagulant therapy, very elderly patients, and others, in whom the toxicity of intensive chemotherapy is often unacceptable. For these cases, arsenic trioxide (ATO) has recently emerged as a suitable alternative.

Unlike induction therapy, there is not the same degree of consensus on the most appropriate consolidation therapy, except for giving at least two or three cycles of anthracycline-based chemotherapy. Although the antileukemic benefit provided by the addition of ATRA to consolidation therapy has not been demonstrated in randomized studies, historical comparisons of consecutive studies carried out separately by the GIMEMA and PETHEMA groups suggest that the combination of ATRA and chemotherapy for consolidation may also contribute to improving therapeutic results in APL. Another interesting issue addressed in the aforementioned GIMEMA and PETHEMA studies [4, 5] was the design of risk-adapted approaches to administer distinct treatment intensities for consolidation based on pre-defined risk of relapse. According to these studies, this strategy seems a suitable approach to minimize therapy-related morbidity and mortality while maintaining the potential of cure for each relapse-risk group. It is remarkable that both studies reported low toxicity, high degree of compliance and high antileukemic efficacy using ATRA combined with anthacycline monochemotherapy, especially in low-and intermediate-risk patients with APL.

Using monochemotherapy with anthracyclines for both induction and consolidation therapy, which led to a significant reduction in treatment-related toxicity during the consolidation phase and a high degree of compliance, the LPA96 study of the PETHEMA group reported outcome results similar to those obtained in other major studies using anthracycline-based chemotherapy combinations. In November 1999, aiming to improve the antileukemic efficacy in patients with increased relapse risk, the PETHEMA started the trial LPA99 based on a risk-adapted strategy. The results obtained in the first 426 consecutive patients with newly diagnosed PML/RAR positive APL who were enrolled in these two consecutive studies (LPA96 and LPA99) were recently reported in Blood [5] . This study, which was recently updated including a significantly higher number of patients and longer follow-up than in the first report, shows that combining ATRA with anthracycline monochemotherapy for induction and consolidation, followed by ATRA and low dose methotrexate and mercaptopurine for maintenance therapy, results in extremely high antileukemic efficacy, moderate toxicity and a high degree of compliance in patients with APL. The novel addition of ATRA to consolidation therapy, combined with a moderate increase in the dose of anthracycline for intermediate-and high-risk patients, resulted in higher antileukemic activity with no additional severe toxicity.

Overall, 735 patients, ranging from 2 to 83 years of age, were eligible for AIDA induction from November 1996 to June 2005. Remission induction rates were similar in both LPA96 and LPA99 trials, 89% and 91%, respectively. Induction failures were mainly due to death during remission, confirming the virtual absence of drug resistance. It should be noted that the only 4 cases labeled as resistant leukemia were all evaluated too early for response, between the days 19 and 33 after completion chemotherapy. Today, it is well known that a proportion of patients need up to 40 or 50 days to complete terminal differentiation of blasts. The two major causes of failure were bleeding and infection, accounting for around 5% and 3%, respectively. No impact was observed in the mortality rate due to hemorrhage according to the use of antifibrinolytic prophylaxis with tranexamic acid nor in the morbidity and mortality rate associated to the retinoic acid syndrome according to the use of prednisone prophylaxis.

Regarding post-remission outcome of patients treated with the protocol LPA99, 2% of patients died in remission. However, mortality rate in patients younger than 60 years was 0.6%, whereas it was significantly increased in elderly patients (patients 60-70 years, 5.2%; patients older than 70 years, 19.2%). The 5-year disease-free (DFS) and relapse-free survival (RFS) is 89% and 91%, respectively, whereas cumulative incidence of relapse (CIR) is 9%. These estimates still show significant differences according to WBC count. DFS and RFS at 5-years in patients with less than 10 thousand leukocytes were 93% and 95%, respectively, with a CIR of 5%. In contrast, patients with more than 10 thousand leukocytes at presentation have a CIR at the same time point of 22%.

In conclusion, this updated analysis on a large series of patients with APL confirms that a risk-adapted strategy combining ATRA and anthracycline monochemotherapy provides a high antileukemic efficacy coupled with low toxicity and high degree of compliance. This improved antileukemic efficacy was certainly caused by the modified consolidation therapy. Although it is unclear which part of the reinforced consolidation therapy (ATRA or chemotherapy or both) may have led to the impact observed in the outcome, it is likely that the addition of ATRA has had a significant role. Based on these results and those recently reported by the GIMEMA Group, [4] we believe that the current consensus on the simultaneous administration of ATRA and chemotherapy for induction and maintenance therapy of APL could be extended to the consolidation phase. In addition, future clinical investigations should focus on developing new therapeutic approaches to decrease the relapse rate in high-risk patients with hyperleukocytosis at presentation and progressively decreasing treatment intensity for the remaining patients. Keeping in mind these thoughts, the PETHEMA and HOVON groups have designed and recently started a new risk-adapted protocol (LPA2005) with the following mainstays: 1) Low-and intermediate-risk patients will be treated with a combination of ATRA and anthracycline monochemotherapy for induction and also for consolidation, followed by ATRA and low dose chemotherapy (methotrexate and mercaptopurine) for maintenance therapy. The addition of ATRA in consolidation therapy for low-risk patients and a dose reduction of mitoxantrone for low-and intermediate-risk patients are the changes proposed for the currently ongoing study (LPA2005) in comparison with the previous one (LPA99); and 2) The still unsatisfactory relapse rate observed in high-risk patients has induced to reinforce consolidation chemotherapy with the addition of ara-C to the idarubicin courses for patients younger than 60 years. This option was based on the results recently reported by the GIMEMA4 and the European APL groups that suggest a role of ara-C in this setting.

The incidence of AML spans the entire age spectrum but is highest in adults greater than 60 years of age. Large numbers of randomized trials have been performed in patients with AML including comparative evaluations of different doses and types of chemotherapeutic agents (in particular, different types of anthracyclines and different doses and schedules of administration of ara-c), the use of hematopoietic growth factors, stem cell transplantation in first remission and modulation of various mechanisms of intrinsic drug resistance Some of these trials have, in fact, changed the standard care of the disease in younger patients, but unfortunately, most have failed to improve remission rates and overall survival in both older and younger patients. Clinical trials in patients 60 years and older have shown remarkably consistent poor results, with complete remission (CR) rates of~50%, with only 10% of patients surviving >2-3 years [1] [2] [3] [4] [5] [6] [7] [8] . In fact, it is arguable that the prognosis of older patients with AML has not changed in the last 15 years and that these estimates of outcome represent a "best case" scenario, since patients with significant co-morbid conditions are not eligible for most clinical trials. Therefore, it is imperative that new therapies to improve on these results be evaluated as efficiently as possible.

There are multiple well accepted explanations for the inferior outcome seen in AML in older patients including: evolution from myelodysplastic syndromes even in some patients with apparently "de novo" AML; a high incidence of chromosomal abnormalities associated with drug resistance; over-expression by the leukemia cells of drug resistance proteins such as p-glycoprotein with an indication that the AML frequently arises in more primitive hematopoietic stem cells; poorer tolerance of the side effects of therapy; alterations in drug metabolism related to subtle or more clinically obvious abnormalities in renal or hepatic function; impaired marrow reserve with delays in count recovery following induction and consolidation chemotherapy.

Indeed, a number of experts have suggested that induction therapy be avoided in more elderly (>70 years) individuals with a reliance on supportive care with hydroxyurea and transfusions. While this may be appropriate in frail individuals with other severe medical problems, this approach affords no possibility of longer term survival. Others have suggested that new investigational therapies, including non-cytotoxics, be used as initial treatment in older patients, arguing that it is "difficult to do worse" than with the current standard therapies. However, although the CR rate with standard chemotherapy is less than ideal, these remissions can last for many months and occasionally years, during which time the patient is quite functional with a normal quality of life. The potential loss of this benefit must be considered before deciding to evaluate therapy with as yet unproved response rates.

AML in older patients is biologically and clinically heterogeneous. Some patients, perhaps more commonly those evolving from MDS, have slowly progressive disease with stable to slowly worsening cytopenias, providing an opportunity for observation without intervention or perhaps permitting the use of new non-cytotoxics with more benign side effect profiles. Recent studies with farnesyl transferase inhibitors and other agents being evaluated in higher IPSS risk groups of MDS are examples of this approach. But, it must be kept in mind that patients with more "proliferative" AML and cytopenias die very rapidly with generally only one chance to achieve remission and agents which act very slowly (or have low response rates), will likely compromise outcome in such patients.

Currently, there is no shortage of new agents which could and should be evaluated in AML. In addition to a continued supply of cytotoxic drugs, there will be large numbers of anti-angiogenesis compounds, immune modulators, signal transduction inhibitors (either with specific or more generic enzymatic targets), as well as new and less acutely toxic approaches to stem cell transplant. Many of the non-cytotoxic therapeutic approaches have the advantage of oral administration with potentially less toxicity. There is little information about the preferred dose and schedule of these agents and the optimal manner (for example, concurrently or sequentially) in which they can be combined with standard or investigational chemotherapies.

New therapeutic approaches should focus both on increasing remission rates as well as prolonging remission and enhancing the cure fraction of such patients. Many AML studies have focused on older patients because of the large numbers of such patients as well as the feeling that the overall results of therapy are so poor that it would be possible to identify truly active agents very rapidly because differences with historical or randomized controls would be obvious.

Because remissions are generally brief, evaluations of new drugs used post remission is one possible strategy. There are a number of both practical and biologic issues complicating the conduct of such trials: • Evaluation of post-remission manipulations is made more difficult by the low complete response rate, so that less than 50% of patients initially entered on trial are eligible for post-remission treatment. In addition, many patients are not candidates for subsequent chemotherapy because of compromised organ function from toxicities encountered during induction, or because of slow or incomplete count recovery. • Furthermore, many older individuals decline post-remission treatment, preferring to spend their remaining time outside of the hospital, as far from aggressive medical ministrations as possible. Thus, randomized studies of new therapies introduced post-remission need larger numbers of patients to account for this drop-off in patients as the study progresses. This represents a major issue in the United States since only a small fraction of such patients are captured for clinical trials.

In addition, AML in older individuals is biologically heterogeneous. Some therapies might be appropriate only for certain AML subtypes and positive effects can be missed when tested in the overall AML population. This may be particularly true for newer targeted therapies. A focus on patients with highly resistant disease represents a high hurdle for new therapies and treatments. Smaller benefits which could be of value to other patient groups, could be missed by studying only patients in very poor prognostic groups.

Randomized trials are usually required to prove benefit from newer treatments. If an agent can be safely added to the usual dose of conventional therapy, it might be most efficient to utilize the new therapy in both induction and consolidation, thereby perhaps maximizing the chance to detect antileukemic activity. Possible study designs for trials of new post-remission therapies are shown in Table 1 where conventional therapy might refer to a few courses of low dose ara-c which results in a median remission durations of approximately eight months and less than 10% long-term disease free survival. This is slightly better than observation without treatment which produces very few if any long-term disease free survivors and shorter CR durations. The choice among the various randomized approaches would be influenced by the unique features of the agent being tested.

Such randomized trials usually require large numbers of patients and take years to conduct. For example, the Medical Research Council recently reported on the results of a randomized trial involving >1600 patients, which unfortunately failed to demonstrate any advantage to a variety of different induction and post remission manipulations in older AML patients [8] . In the absence of a compelling hypothesis for a phase III trial (and I do not think there are many such "burning" questions which need evaluation), an alternative and more appealing approach could be the serial conduct of smaller phase II trials, designed to identify promising new leads for subsequent evaluation in larger studies. Although there is a possibility of "false negatives" with smaller studies with the risk of abandoning an approach which might actually be helpful, this is balanced by the potential for more rapid identification of true "positives" which can then be studied further with modifications of dose and schedule and most importantly, in vitro molecular or pharmacologic studies designed to identify patients most likely to benefit from the new treatment. Although "false positives" are also an issue, the imperative to more rapidly evaluate new approaches, still makes this approach attractive.

The rapid conduct of phase II trials, designed either as sequential single arm studies or as randomized phase II efforts will require newer administrative approaches to seamlessly "jump" from one study to another with as little down time as possible. Prewritten "master" phase II protocols could facilitate this process which will require a commitment and new mindset from investigators. Another practical issue is that many compounds of interest are owned by pharmaceutical companies and the groups conducting such trials must demonstrate that they can be done efficiently and more credibly than other alternatives so that companies become more willing than they have been in the past, to allow evaluation of their agents by "outside" groups.

Acute myeloid leukemia in the older patient is a common and important, disease which is relatively resistant to current therapies. There will be many compounds available for evaluation in upcoming years and it is desirable that such studies be conducted using the most efficient and informative designs to rapidly identify therapies which lengthen survival and increase the fraction of patients who are cured. 

Based on recent advantages in experimental and clinical research, acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) have been recognized as a heterogenous group of malignancies. Accordingly, different subgroups could be defined, showing a broad variety in terms of prognosis, ranging from rather sensitive diseases with a reasonable chance for cure by conventional chemotherapy, to refractory cases with a median survival of few months. Increased risk can defined (1) at time of diagnosis and (2) during the course of the disease. At diagnosis, the karyotype of the malignant clone is the best established risk factor1-3. Although some difference exist among the definitions of prognostic cytogenetic subgroups, all research teams have identified t(15;17), inv16 and t(8;21) as favorable aberrations, whereas monosomy 5 and 7, del 7q, del 5q (in AML, not in MDS), and a komplex aberrant karyotyp (i.e. 3 or more aberrations) are associated with a dismal outcome. A history of MDS or of chemo/radiotherapy prior to the diagnosis of AML represents another adverse factors, as does progressive MDS at a stage RAEB I or II. During the course of the disease, increased risk can be defined by failure or delayed response to induction chemotherapy4,5, early relapse after a remission duration of less then 6-12 months, second or higher relapse and relapse following high-dose therapy and autologous stem cell transplantation. 6 In the light of unsatisfying results with conventional therapy, allogeneic stem cell transplantation (SCT) is the recommended treatment for high risk AML and MDS.7-9 However, due to relapse and treatment-related complications, long term survival is rare.10 Particularly in elderly patients, patients with MDS and secondary AML, and patients with advanced disease, non-relapse mortality (NRM) may reach >70%.11-14 Therefore, the development of new transplantation strategies is warranted in order to find a balance between antileukemic activity and treatment-associated toxicity. (15) During recent years, the role of high-dose chemo/radiation therapy for the elimination of leukemia in allogeneic SCT has been questioned. Instead, the graft-versus-leukemia (GvL) effect appears to play a major role. GvL reactions are known from animal studies (16) and clinical trials (17) . In AML, evidence for a GvL effect comes from the lower relapse rates and improved progression-free survival following allogeneic as compared to autologous HSCT, (18) and from the higher risk of relapse in patients who received a T-cell depleted transplant or did not develop graft-versus-host disease (GvHD) (17) .

Reduced intensity conditioning (RIC) regimens have been advocated to reduce transplantation-associated toxicity in elderly or medically unfit patients. Initial studies impressively showed the feasibility of RIC transplantations in a variety of hematological disorder and in patients of different stages of their disease (19) (20) (21) (22) (23) . However, so far it is uncertain, to what extend the conditioning regimen can be reduced in a given patient without jeopardizing the efficacy of the whole procedure. In particular in progressive leukemia, concerns have been risen, that RIC may not sufficiently control the disease to allow a GvL effect to pursue. The intensity of the preparative regimen has been shown to directly influence relapse incidence and leukemia-free survival after allografting for AML and MDS, (24) and disappointing results have been reported from RIC transplants in advanced disease (25) . The group from Seattle has published a study on non-myeloablative transplantation using 2 Gy TBI +/-fludarabine for patients with AML in first complete remission (CR1), who were not eligible for standard conditioning (26) . However, relapse rate was 41%, and the 1-year progression-free survival was 42% only.

In an attempt to increase the safety of allogeneic SCT while maintaining the antileukemic efficay, our group has designed a protocol, that intended to separate the two main aspects of the preparative regimen, i.e. cytoreduction and immunosuppression. First, intensive chemotherapy was applied for cytoreduction, using fludarabine (30mg/m2), high-dose AraC (2g/m2) and amsacrine (100 mg/m2) from day -12 to -9 (FLAMSA regimen). Similar protocols had been effective in high-risk AML and advanced CML (27) . Instead of anthracyclins, amsacrine was introduced. This drug has shown to be similarly effective in poor prognosis AML, while being less cardiotoxic (28) . AML patients have regularly been treated with considerable doses of anthracyclins before transplantation. Therefore, the use of a different drug seemed also reasonable to prevent chemoresistance. Chemotherapy aimed at targeting rapidly proliferating malignant cells in order to reduce the leukemia burden before RIC and SCT. It was followed by three days of rest, allowing time, in particular to the patient's intestinal mucosa, to recover from acute toxicity. Then, RIC consisted of 4Gy total body irradiation (TBI) on day -5, cyclophosphamide (40mg/kg with HLA-id sibling, 60 mg/kg for unrelated or mismatched donors) on days -4 and -3, and rabbit antithymococyte globulin (ATG; 10 mg/kg for HLA-id sibling, 20 m/kg for unrelated or mismatched donors) from day -4 until day -2. For transplantation, G-CSF mobilized peripheral blood stem cells (PBSC) were preferred, bone marrow (BM) was accepted at the donor's preference. GvHD prophylaxis consisted of cyclosporine A (CyA) from day -1, and mycophenolat mofetil (MMF, 2x15 mg/kg), starting from day 0 In the absence of GvHD, MMF was discontinued by day +50, CyA was tapered from day +60 to +90. To increase the allogeneic GvL effect, patients received prophylactic donor lymphocyte transfusions (pDLT), if they were in CR without evidence of GvHD at day +120 or 30 days after discontinuation of immunosuppression. The initial dose was 1x106 CD3+ cells/kg; it could be increased to 5x106 CD3+ cells/kg in patients without a history of aGvHD. In the absence of GvHD, pDLT was repeated up to 3x, using escalating cell doses (5-10 fold increase/transfusion) at 4-6 weeks' intervals. The protocol is summarized in Figure 1 .

The protocol was first evaluated in a prospective pilot trial of 75 consecutive patients at the Ludwig-Maximilians-University Hospital of Munich (29) . In contrast to other trials investigating modern RIC regimen, (30) (31) (32) inclusion criteria were not based on medical contraindications against standard conditioning, but on an increased risk disease, as defined by one or more of the following criteria: (1) primary or secondary refractory leukemia (as defined by persisting disease following 1 course of high-dose AraC), (2) delayed response to induction chemotherapy (3) The regimen proved to be myeloablative in all cases. Neutrophil engraftment occurred at a median of 14 days from transplantation, and donor chimerism of > 90% was achieved at day +30 in the vast majority of patients in the bone marrow and the peripheral CD3+ compartment.. 66 patients achieved a CR, 5 died in aplasia, and 4 gad persisting leukemia at time of hematopoietic reconstitution. The overall non-relapse mortality (NRM), including deaths related to concomitant disease, was 20% at day +100 and 33% at one year. Patients with an HLAidentical family donor, and PBSC recipients receiving higher CD34+ cell doses had less NRM. Leukemic death occurred in 17 patients: 4 died from refractory leukemia early after transplantation, 13 died from relapse. Having an HLAidentical family donor was a risk factor for leukemic death After a median follow up of 31.5 months, overall survival (OAS) and disease free survival (DFS) were 42 and 40% . In a multivariate analysis, a higher number of CD34+ cells in PBSC recipients was the only pre-transplant variable being associated with better survival. After transplantation, acute and chronic GvHD had a significant influence on outcome: Severe forms were associated with high NRM and were deleterious for outcome, whereas patients with mild GvHD did significantly better. Most interestingly, neither cytogenetic subgroups, nor the stage of the disease at transplantation had a significant influence on outcome. In particular, patients with complex cytogenetic aberrations, and patients with chemorefractory leukemia showed a 2 year OAS of 42% and 52%, respectively, thereby equalizing the outcome of the entire cohort.

Prophylactic transfusion of donor lymphocytes (pDLT) could be given to 12 patients only, representing 16% of the entire cohort, an 24% of the patients alive at day +120, which was the earliest date for pDLT. In contrast, early relapse, GvHD or ungoing immunosuppression prevented pDLT in the majority of the patients. The outcome of pDLT recipients was very promising, with 11/12 patients (92%) being alive at three years from transplantation. However, it might well be, that by using very strict inclusion criteria for pDLT, a postitive subgroup has been selected, that anyway would have shown an excellent outcome.

Therefore, the role of pDLT for the antileukemic effect of the entire treatment remains to be defined.

Consequently, the FLAMSA-RIC protocol was adopted by several transplant centers. Based on their data, an analysis of 174 transplants following FLAMSA-RIC could be presented at the EBMT meeting in March 2005 in Prague (33) . Hence, the results of the pilot trial were confirmed, with the entire cohort of high risk patients showing an OAS/DFS of 53%/48% at one, 44%/42% at two, and 39/38% at four years from transplantation ( Figure 2 ). Multivariate analysis revealed a dose of CD34+ cells in the graft higher than 8 x 106/kg as a major factor for improved outcome (Figure 3 ). The beneficial role for a mild acute GvHD was confirmed as well (Figure 4) GvL effect even in these highly proliferative disorders. This is also supported by the excellent results achieved in 31 patients who received pDLT ( Figure 5 ).

Reduced conditioning regimen directed against AML and MDS have been developed by several other groups (30) (31) (32) (34) (35) (36) . As with the FLAMSA-RIC regimen, the inclusion of fludarabine is a common feature of all protocols. Fludarabin was combined with melphalan, busulfan given I,v. or p.o., or TBI. A formal comparison among these protocols is difficult due to strong imbalances with respect to patients' selection. However, impressively low toxicities and nonrelapse mortality rates have been reported. Whereas most studies were too small for subgroup analysis, de Lima et al. 35 and Stelljes et al.36 analyzed the influence of the disease status at time of transplantation. In both studies, results were excellent for patients transplanted in remission, but were clearly inferior in patients with active or refractory disease. These results suggest, that in the future, modern transplantation regimen will have to be evaluated in precisely defined patient subgroups in order to define the optimal, risk adapted approach for each individual patient. In Germany, the protocol proposed by Stelljes et al. is currently under investigation in a prospective, randomized multicenter trial for allogeneic transplantation for AML in first CR.

The follow the idea to specifically design risk adapted transplant concepts, two studies were initiated to evaluate the role of the FLAMSA-RIC regimen in defined patient groups, whose results within the pilot trial had suggested a particular benefit. First, we studied the outcome of 91 patients with AML that was classified as refractory to conventional chemotherapy, based on established criteria (6, 37, 38) . Accordingly, refractoriness was defined by (1) primary induction failure (PIF) after two cycles of induction therapy, (2) a duration of first remission of less than 6 months, (3) second or higher relapse, and (4) relapse that is refractory to re-induction therapy containing high-dose AraC. Ninety-one patients fulfilling these criteria were transplanted in eight centers in Germany and Austria.39 An unfavorable karyotype was found in 49%. 37 patients had an HLA-identical family donor, 54 had a mismatched or unrelated donor. Median time from diagnosis to SCT was 194 (range: 73-599) days, and median follow up was 24.5 months.

Overall survival (OAS) was 53%, 37% and 30% at 1, 2, and 4 years from SCT, leukemia free survival (LFS) was 47%, 37% and 30% at 1, 2 and 4 years. Patients with PIF did significantly better than patients with early relapse (LFS at 2 years: 59% vs. 22%, p=0.003, log rank). In multivariate analysis, a higher number of courses of chemotherapy before SCT was the most important risk factor for inferior outcome (p=0.02, HR 2.26 for OAS and p=0.016, HR 2.34, for LFS). Cumulative relapse incidence at 1 year after SCT was 48%, whereas TRM was only 19% at 1 and 25% at 2 years. These results compared favorably with the results of published studies on refractory AML using standard conditioning regimen (15) . While the incidence of relapse and leukemic death is still considerably high, major progress has been made with respect to nonrelapse mortality, Therefore, the sequential strategy of intensive chemotherapy for cytoreduction and reduced conditioning applied within the FLAMSA-RIC protocol seems to show a way for reducing the toxicity of allogeneic SCT without significant loss of antileukemic activity.

The second subgroup currently under investigation consists of patients with complex aberrant karyotype in their leukemic blasts. Within the German AML Cooperative Group (AMLCG) study 1992, the outcome of these patients was dismal, showing a median OAS from diagnosis of 6 months only.40 Standard chemotherapy does not seem to be a real option in the majority of these patients. As mentioned above, OAS was 42% at two years from allogeneic transplantation following FLAMSA-RIC. Therefore, a prospective multicenter trial is actually ongoing within the AMLCG, evaluating the feasibility and efficacy of an early (i.e. after one or two induction cycles) allogeneic SCT following FLAMSA-RIC. The study was started in October 2004 and has so far recruited 50% of planned patients.

The data summarized here, as well as the results of other trials investigating modern conditioning regimen, have shown that major steps ahead have been done in the field of allogeneic transplantation for AML and MDS. It will have to be answered by future trials, which one out of the available protocols will be the best in a defined clinical situation. FLAMSA-RIC might be an option for patients with the poorest prognosis, i.e. with refractory disease or complex cytogenetic aberrations. In contrast, less aggressive regimen might be preferable in earlier stages. 

The most important prognostic factors in AML with respect to survival are age and cytogenetics. The incidence of distinct karyotype abnormalities varies with age. While in younger adults the favorable chromosome abnormalities and balanced rearrangements are more frequent, unfavorable cytogenetics, especially complex aberrant karyotypes and other unbalanced karyotype abnormalities predominate in elderly patients. Several studies showed an independent impact of age and cytogenetics on clinical outcome, demonstrating that the poorer outcome in AML of the elderly is not solely due to the more unfavorable pattern of cytogenetic aberrations. The impact of age within distinct cytogenetic subgroups as well as the impact of cytogenetics within age groups were studied in detail and revealed that 1. both age and cytogenetics are independent prognostic parameters in AML, 2. the influence of age on outcome increases beyond 50 yrs, 3. cytogenetics show an independent effect on survival in all age groups.

In AML several pretherapeutic parameters such as cytogenetics, age, WBC, LDH, and the history of the disease (occurring de novo vs. after an antecedent hematological disorder vs. therapy-related) were shown to be of prognostic importance [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] . The early assessment of response to therapy as measured by the morphologic parameter early blast clearance in the bone marrow represents an in vivo assessment of chemosensitivity and is also a powerful tool to delineate the prognosis in individual patients [12] . Although patients above 65 years comprised 63% of all leukemia cases only 27% of patients in leukemia trials were over 65 years of age [13] . Despite the fact that patients above the age of 60 are usually underrepresented all studies demonstrate an inferior outcome in the elderly. Several aspects contribute to the correlation of a worse prognosis with increasing age in AML. These can be divided into a) individual patient factors like comorbidity, b) biological leukemia factors such as higher proportion of unfavorable karyotypes [14] [15] [16] and higher frequency of secondary AML as well as c) treatment factors such as less intensive treatment regimens. Results of several studies revealed that differences in the distribution of cytogenetic risk groups influence but do not fully explain the more unfavorable outcome with increasing age. Although cytogenetics is of prognostic impact in younger and elderly patients the survival of patients 60 years and older is inferior to younger patients in each cytogenetic category with exception of APL. It has to be considered that differences in treatment protocol may be at least partly responsible for the more unfavourable outcome in patients 60 years and older as they usually receive less intensive treatment compared to younger patients.

Overall prognosis of patients with complex aberrant karyotype is very poor, only patients younger than 50 years had a slightly better outcome than the other age groups. This may be due to a higher rate of allogeneic bone marrow transplantation in this cohort.

In conclusion, both age and karyotype of the leukemic blasts have an independent prognostic impact in adult de novo AML. Age has no major impact on prognosis up to the age of 49 years. Beyond this threshold the influence of age on outcome increases. In all age groups separation of patients on the basis of the karyotype resulted in prognostically different subgroups [17] . Therefore, cytogenetics is mandatory in all age groups to allow risk adapted treatment approaches and outcome prediction. Recently, a number of submicroscopic genetic lesions with prognostic impact have been identified in patients with AML and a normal karyotype. These data show the genetical heterogeneity of this cytogenetic subgroup and provides first clues to understand the biology of AML in these patients. In the following the four most frequently altered genes: NPM1, FLT3, CEBPA and MLL are discussed. These genes can be altered in theit physiological function by rearrangements, mutations or overexpression and can serve as prognostic markers for prognosis, detection of minimal residual disease (MRD) and also for the development of targeted therapies.

Very recently, Falini et al. have shown that an abnormal cytoplasmatic localization of the NPM-Protein can be found in about 35% of all patients with primary acute myelogenous leukemia (AML), but not in secondary AML and other hematopoietic or extrahematopoietic neoplasms other than AML (1). Cytoplasmatic NPM staining was associated with a normal karyotype, and responsiveness to induction chemotherapy, but not with recurrent genetic abnormalities. In patients with a normal karyotype, cytoplasmatic NPM was found in 62%.

The cytoplasmic localization of NPM was caused by mutations of the NPM gene that alter the nuclear signaling motif. The original data of Falini et al. have been reproduced by a number of other groups. According to a recent study in 401 patients with a normal karyotype FLT3-ITD mutations are found in 40% of patients with NPM1 mutations and represent the most frequent additional genetic alteration in patients with a mutated NPM gene (2) .

Interestingly, the favorable impact of NPM1 mutations on survival was clearly seen in the group of normal-karyotype AML without FLT3-LM. This favourable effect was lost in the presence of a FLT3-ITD (2).

The physiological function of NPM is complex and includes regulation of DNA integrity and inhibition of cell proliferation by interaction with p19ARF. These data point to a putative tumor-suppressive function of NPM and loss of its function is likely to cooperate with FLT3-ITD to initiate or maintain the leukemic phenotype in AML. Animal models have established that NPM is essential for embryonic development and the maintenance of genomic stability (3). Moreover, NPM haploinsufficiency accelerates oncogenesis in vitro and in vivo model systems. Mice expressing a hypomorphic NPM allele develop a haematological syndrome similar to human MDS and point to a central role of NPM in the pathogenesis of AML.

These data clearly show that NPM mutations represent the most common submicrosopic alterations in patients with a normal karyotype and have a profound diagnostic and prognostic impact. Analyses from our and other groups have shown that the frequency of FLT3-ITD mutations differs significantly in cytogenetic subgroups of AML patients and is highest in patients with a normal karyotype and in patients carrying a t (15;17) translocation. In contrast, the FLT3-LM are rarely found in patients with a complex karyotype and CBF-leukemias (CBF-MYH11 and AML1-ETO). In all cytogenetic subgroups, the presence of a FLT3-LM represents a negative prognostic risk factor for the overall and event free survival. Furthermore, the loss/deletion of the residual FLT3-WT allele represents an additional negative prognostic factor in FLT3-LM positive AML. These findings are supported by data from our group showing that deletions of the FLT3-WT allele are more frequently found in patients at relapse as compared to patients at initial diagnosis suggesting that these genetic alterations are associated with disease progression.

Genetically, FLT3-LM are heterogeneous and consist of internal tandem duplications (ITD) of 6-30 amino acids (AA) in most patients. These mutations result in an elongated FLT3 protein with constitutive protein tyrosine kinase (PTK) activity. Recent structural analyses of the Ephb2 family of RTK show that the JM domain forms an inhibitory loop, which interacts with the catalytic domain of the kinase. Mutations in the JM region probably interfere with the inhibitory activity of this domain resulting in a constitutive catalytic activity of the kinase. Constitutively active FLT3 mutants have transforming potential in IL-3 dependent cells and constitutively activate the STAT5 and MAPK pathways (4). The transforming capacity of active FLT3 mutants has been confirmed in animal models either by overexpression of FLT3-LM in the BMT model or in transgenic animals expressing an active TEL-FLT3 fusion protein. Both models show that the constitutively active FLT3 can induce a myeloproliferative syndrome in vivo and underline the potential transforming activity of these mutants in AML. However, these data also show that active FLT3 mutations alone are not sufficient to induce an AML phenotype in vivo and support the concept of a multistep pathogenesis in AML. Very recently, Gilliland et al proposed a model that distinguishes two types of mutations in AML. According to this model, type I mutations represent genetic alterations which induce a proproliferative and anti-apoptotic signal (e.g. gain of function mutations of PTK and ras) whereas type II mutations interfere with differentiation and often involve myeloid transcription factors (loss of function mutations).

This model is supported by experimental data indicating that AML1-ETO and FLT3.ITD mutations, two of the most frequent genetic alterations in AML, are both insufficient on their own to cause leukemia in animal models. We could recently show that AML1-ETO collaborates with FLT3-LM in inducing acute leukemia in a murine BM transplantation model (5) .

Mutations of the CEBPA Gene C/EBP is a member of the bZIP family and consists of N-terminal transactivating domains, a basic region necessary for specific DNA sequence binding, and a leucine-zipper region necessary for dimerization at the C-terminal end. In vivo and in vitro studies have clearly shown that C/EBP plays an important role in the regulation of myeloid differentiation. Conditional expression of C/EBP triggers neutrophilic differentiation while C/EBP knockout mice exhibit an early block in maturation.

Mutations in the CEBPA gene can be detected in 7% to 15% of patients with acute myeloid leukemia, mostly in the FAB AML M1 or M2 subtype and in those with normal karyotype. These mutations largely fall into two major categories: one prevents C/EBP DNA binding via alteration of its COOH-terminal bZIP domain, and the other disrupts translation of the C/EBP NH2 terminus. The latter mutation results in a formation of a 30-kDa C/EBPp30 isoform. This 30-kDa isoform exerts a dominant negative effect reduces wild-type C/EBP activity by inhibiting its DNA binding.

Interestingly, several groups have shown that CEBPA-mutations are associated with a favourable prognosis. In one large study 36/256 (15%) patients with a normal karyotype carried CEBPA-mutations and had a significantly longer remission duration compared to patients with CEBPA-WT (6) . An alternative mechanism of C/EBP inactivation has been described by Pabst et al. who found that the AML1-ETO fusion protein encoded by the t(8;21) suppressed C/EBP transcription (7) .

These data show that two distinct mechanisms of C/EBP inactivation contribute to a common leukemogenic pathway and provide important insights in the mechanism of the differentiation block in AML.

The mixed lineage leukemia (MLL) gene represents the human homologue of the Drosophila trithorax gene ands is located at chromosome band 11q23. MLL gene translocations, deletions, and duplications can be found in patients with AML and most often result in gene fusions between the 5´-end of the MLL gene and the 3´-end of a partner gene.

In addition, submicroscopic rearrangements of the MLL gene are the partial tandem duplication of MLL (MLL-PTD). At the level of transcription MLL-PTD result in a unique in-frame fusion of exons 11 or 12 upstream of exon 5. Functional data support the hypothesis that MLL-PTD induce a loss of MLL WT function via monoallelic repression thereby contributing to the leukemic phenotype by the remaining mutant allele. In line with these findings are mouse models of MLL haploinsufficiency. MLL-WT/-mice have a defect in their hematopoietic compartment, consistent with a gene dosage effect and suggest that two WT MLL genes are required for normal hematopoiesis MLL-PTD are frequently found in adult de novo AML and are associated with a worse prognosis (ie, shorter duration of remission) when compared with normal karyotype AML without the MLL PTD. In addition, several studies have shown that MLL-PTD are more frequently found in FLT3-ITD positive compared to FLT3-ITD negative patients.

New Approaches for Subclassification of AML with a Normal Karyotype: Gene Expression Profiling Although the established genetic markers NPM, FLT3, CEBPA and MLL have substantially improved the subclassification of AML with a normal karyotype further insights into the molecular heterogeneity are necessary. To find molecular signatures that identify prognostic subgroups microarray gene expression profiling has been used. Two recent studies have adressed this topic: Bullinger et al. could show that AML patients with a normal karyotype can be divided in two subgroups with different overall survival (8) .. Major discriminating variables were the FLT3 mutations status and the FAB subtype. Using supervised learning, an optimal clinical-outcome predictor was constructed, which accurately predicted overall survival among patients in the subgroup of patients with AML with a normal karyotype.

In another study of Valk et al. AML patients with a normal karyotype were grouped into several clusters, three of which accounted for at least 75% of patients (9) . Although no prognostic evaluation was performed in the latter study, these results clearly show that the molecular gene expression signature will help to subclassify the group of patients with normal karyotypes with regard to prognostic and biological relevant parameters.

Since currently available therapy for acute myeloid leukemia is toxic and nonspecific, and particularly in older adults(1) generally ineffective, there has been a search for therapeutic targets analogous to bcr-abl in chronic myeloid leukemia. In contrast to stable phase chronic myeloid leukemia, the pathogenesis of AML is presumed to require multiple genetic derangements or 'hits.' (2) The possibility that inhibiting a critical pathophysiological event could interrupt the leukemogenic process has been an intriguing notion in developmental therapeutics. While some strategies have involved targeting mutated forms of the guanosine residue-binding protein, ras,(3) the most interesting therapeutic target in AML over the last five years has been the transmembrane receptor tyrosine kinase FLT3.

Pre-Clinical Development FLT3 (FMS-like tyrosine kinase) is a transmembrane tyrosine kinase in which ligand binding triggers a pro-proliferative signaling cascade. The FLT3 protein is expressed in 80% of blasts from patients with AML; the expression level has been deemed to be 'high' in 50% of such patients.(4) The critical observation is that an activating mutation of FLT3 can be found in leukemic cells from 30-35% of patients with AML. These mutations fall into two subtypes: 25-30% of AML patients' blasts have a 3-33 amino acid repeat (internal tandem duplication; ITD) in the juxtamembrane region of the molecule. Another 5-10% of AML patients' blasts can be shown to have a point mutation, typically an aspartate to a tyrosine mutation in the 835th codon in the so-called activation loop that resides in the carboxy terminus of the molecule. Each of these mutations confers ligandindependent kinase activity.(4) Constructs carrying either type of mutation are capable of conferring factor-independent growth in factor-dependent leukemic cell lines.(5) Secondly, mice who receive marrow transplants with stem cells carrying an activated FLT3 mutation develop a fatal myeloproliferative disorder. (6) In keeping with the notion that multiple genetic events are required for fullblown leukemogenesis, the leukemic cells from such animals are more mature appearing than would be seen in a typical patient with AML. Additional mutations, such as those transcription factor abnormalities seen in good prognosis subtypes of AML, are required to cooperate with such tyrosine kinase mutations to cause full-blown murine leukemia in model systems. (7) There have been numerous clinical studies in the past decade that have examined the relationship between oncogene mutations and the natural history of AML. Most studies have shown that internal tandem duplication mutations in the FLT3 tyrosine kinase confer adverse prognosis. (8, 9) This is particularly true for AML patients with a normal karyotype. Activating mutations of FLT3 tend to be more common in those with either a normal karyotype, the t(15;17) typical of acute promyelocytic leukemia (APL) or the t(6;9) translocation characteristic of the rare entity of AML with basophilia.(4) It is controversial whether a FLT3 mutation is an independent negative prognostic factor in APL;(10) moreover, studies have failed to show a consistent correlation between activation loop mutations and prognosis.(11) It does appear that loss of the normal allele in conjunction with a FLT3 mutation (hemizygosity) is a particularly ominous prognostic sign. (12) Various strategies to inhibit FLT3 signaling are in development including antibodies (13) and chaperone protein inhibition (14) to decrease the half-life of the molecule, but the greatest interest has been in the small molecule tyrosine kinase inhibitors including SU112428, SU5416, CEP701, MLN518, and PKC412 which have each gone through pre-clinical and early clinical testing. They specifically kill leukemic cell lines transformed with either of the activated constructs of FLT3.(15) Moreover, these agents have been shown to prolong the survival of mice with a fatal activated FLT3-mediated murine myeloproliferative disorder.(16) Each of these molecules inhibits FLT3 in the nanomolar range but they vary in terms of chemical structure, preclinical side effect profile and most notably the spectrum of kinase in which they inhibit. For example, PKC412 not only inhibits the FLT3 tyrosine kinase but it also inhibits ckit, and the serine-threonine protein kinase C. (17) Single Agent Clinical Studies The initial development of these agents has demonstrated that doses producing plasma levels capable of inhibiting FLT3 are tolerable and are associated with some degree of preliminary biological efficacy in AML patients especially those harboring an activating mutation. The first two FLT3 inhibitors studied, SU11248 (18) and SU5416 (19) were developed in AML not based on FLT3 inhibition, but rather because they were potent inhibitors of the c-kit tyrosine kinase involved in leukemic proliferation and/or the VEGFR tyrosine kinase maintaining leukemic survival via malignant cell-stromal cell interactions. A Phase I/II study of CEP701 (20) was restricted to AML patients who carried an activating mutation of FLT3 and who were not deemed to be chemotherapy candidates. Of the 17 patients treated, several experienced a reduction in their peripheral blast counts. The drug was well tolerated but no complete remissions were achieved. A tolerable dose of the multitargeted tyrosine kinase inhibitor PKC412 had been documented in an earlier Phase I trial in solid tumors (21) carried out because it inhibited protein kinase C. A "proof-of-concept" trial in which PKC412 was administered at a dose of 75 mg orally three times daily to 20 patients who had an ITD (17) or activation loop mutation (3) were recently reported. (22) 70% of the patients experienced at least a transient reduction in their peripheral blast count of which half, or 35% overall, achieved a two-log reduction which lasted at least four weeks. One of the 20 patients achieved a response that met all criteria for complete remission except for marrow hypocellularity. Problems included a lack of durable response in most cases, a greater effect on the peripheral blast count then on marrow blast count, an induction of the drug's own metabolism with continued use, and fatal pulmonary toxicity in two patients. PKC412 was administered at other doses (50 mg twice daily orally or 100 mg twice daily orally) to non-chemotherapy candidate AML patients. Preliminary results of this study indicate that, while there were responses in wild-type patients at both doses, responses were more common and more profound in patients with an activating mutation of FLT3.(23) MLN518 has been subjected to two singleagent trials. The first was a Phase I effort in patients with relapsed and refractory and those deemed inappropriate for chemotherapy. The dose limiting toxicity at (525 mg twice daily) was muscle weakness; several patients who achieved a high enough plasma concentration and had a FLT3 mutation achieved a reduction in peripheral blasts. (24) Phase II testing of this compound in a trial restricted to those with an FLT3 ITD mutation showed blast reductions but no CRs. (25) Small Molecule FLT3 Inhibitors in Combination with Chemotherapy Trials employing the small molecule FLT3 inhibitors as single agents, especially in patients whose blasts have shown to have an activating mutation, demonstrated biological activity, but probably not enough clinical activity to merit their use in this fashion. However, more studies in chemotherapy naïve mutant FLT3 patients remain to be done. Perhaps the best way to take advantage of the intrinsic biological activity of these agents is to combine them with other effective drugs. Although there are many interesting preclinical models in which FLT3 inhibitors have been combined with biological agents, the greatest thrust in recent clinical development has been the combination of FLT3 inhibitors with standard chemotherapy.

Preclinical work with in vitro systems demonstrated that certain chemotherapy agents such as daunorubicin are synergistic for killing FLT3dependent leukemic cell lines. Cytarabine is either synergistic or additive in combination with these agents. However, an important finding is the sequence dependence of the effects. Small and colleagues (26) have shown that pretreatment of leukemic cells with the FLT3 inhibitors make chemotherapy less effective, presumptively by causing cell cycle arrest. Moreover, from a clinical standpoint one must ensure that there are no pharmacokinetic or other interactions between chemotherapeutic agents and the FLT3 inhibitors that could affect the safety of co-administering these agents. CEP701, PKC412, and MLN518 are each being tested in combination with chemotherapy in ongoing clinical trials. Patients with relapsed AML whose blasts have an activating mutation of FLT3 are eligible for the Phase II randomized trial of salvage chemotherapy with mitoxatrone/etoposide/ara-C with or without the co-administration of CEP701. (27) Newly diagnosed patients with non-M3 previously untreated AML under the age of 60 are being enrolled onto a Phase I feasibility study of a combination of standard induction chemotherapy and standard high-dose ara-C consolidation chemotherapy with either simultaneous or sequential PKC412. Approximately one-quarter of the first 30 patients in this trial stopped the drug prematurely due to severe or life threatening side effects, or intolerable nausea and vomiting.(28) However, when the dose of PKC412 was changed from 100 mg twice per day to 50 mg twice daily, only 1 out of 19 patients required early termination due to toxicity. (29) Moreover, 11 of 13 patients with an activating mutation of FLT3 who received chemotherapy in combination with PKC412 achieved remission. A Phase I trial in which escalating doses of MLN518 are combined with standard induction chemotherapy in untreated AML patients of all ages has been recently activated.

The development of FLT3 inhibitors as therapeutic agents to AML has proceeded on a relatively rational basis beginning with target identification followed by the demonstration that activating mutations of FLT3 were pathophysiologically relevant in AML. The complexity of the disease and patient population has seemingly precluded their standard use as single agents in AML. However, biological and clinical activity has prompted further development, particularly in conjunction with standard chemotherapy. It now appears that the most widespread use of one or more of the FLT3 inhibitors in development in AML will come in conjunction with chemotherapy. However, randomized Phase III trials of chemotherapy with or without the FLT3 inhibitors will be required. At the moment, one of the major controversies is whether or not such trials should be conducted in selected groups of AML patients with an activating mutation of FLT3 where the greatest activity would be expected, or whether all AML patients should be included in such an evaluation because of the preliminary results showing activity in non FLT3 mutant patients as well as the possibility that the off-target effects of these drugs might also be beneficial.

The great majority of patients with newly diagnosed acute promyelocytic leukemia (APL) are cured of their disease with contemporary therapeutic strategies. Such success has taken place after a series of studies which include all trans-retinoic acid (ATRA) combined with chemotherapy, 1-2 years of maintenance therapy following induction and consolidation, and ATRA in consolidation. (1-4) However, a small percentage of patients relapse. With current therapy, the relapse rate (RR) for low-and intermediate-risk patients at 3-years appears less than 5 percent.(4) For high-risk patients, the 3-year RR is approximately 20 percent. Late relapses have rarely been reported both in the pre-ATRA as well as the ATRA-era.(3, 5-7) Extramedullary relapses have been suggested by anecdotal reports, but not confirmed by one large study. (8) (9) (10) (11) (12) (13) Important prognostic factors include age > 55-60 years and white blood cell count > 10,000/l. CD56 expression has been suggested as a negative prognostic factor, but not confirmed by all studies. (14, 15) Internal tandem duplications of the FLT3 gene may be associated with an inferior prognosis. (16) (17) (18) (19) (20) A major focus of current therapy is in minimizing chemotherapy in low-and intermediate-risk patients, and identifying new strategies for those at high-risk of relapse. A number of new strategies for high-risk patients are under investigation.

In North American Intergroup Protocol 0129, patients with newly diagnosed APL were randomized to ATRA or chemotherapy (daunorubicin plus cytarabine) for induction. Patients then received two courses of consolidation (daunorubicin plus cytarabine in standard doses followed by high-dose cytarabine plus two days of daunorubicin). Subsequently, patients remaining in complete remission (CR) were randomized to 1 year of daily ATRA maintenance or observation. (3) Patients who were randomized to receive ATRA for induction and ATRA for maintenance had a 5-year disease-free survival (DFS) of 74%. Patients who received ATRA for either induction or for maintenance had a DFS or approximately 50%. In contrast, two recent trials did not show a benefit for maintenance therapy with either ATRA or chemotherapy among patients rendered molecularly negative with intensive consolidation therapy. (21-23) The cumulative doses of anthracyclines given in the North American Intergroup trial were lower than those administered in the trials conducted by the GIMEMA and the JALSG.

Hematopoietic Stem Cell Transplantation for Patients with Advanced Disease Several studies report successful outcome of patients with APL in second CR undergoing autologous HSCT, with reinfusion of molecularly negative cells. (24) (25) (26) (27) In a study from Europe, among 50 patients undergoing autologous HSCT in second CR, the 7-year RFS was 79% with a transplant-related mortality (TRM) rate of only 6%. (27) Among the 28 patients who underwent autologous HSCT with molecularly negative cells collected before transplant, only three patients (11%) relapsed. The TRM, for those patients undergoing allogeneic HSCT was 39%. Therefore, the OS was significantly better among patients undergoing autologous HSCT compared to that observed for patients undergoing allogeneic HSCT. Among 17 patients with advanced APL in either second molecular CR or with persistent molecular disease undergoing matched sibling allogeneic HSCT, DFS was 46% with a non-relapse mortality rate of 32%. (28) Preliminary data from the Center for Blood and Marrow Transplant Research (CIBMTR) show excellent outcome after both autologous HSCT and allogeneic HSCT for patients in first CR, including those with high-risk disease.

Several studies have suggested that intermediate-or high-dose cytarabine may be useful in preventing relapse in high-risk patients. In a report from the German AML Cooperative Group, the presence of a high-WBC ( 5,000/l) was not a risk factor for relapse, when high-dose cytarabine was given in induction (3 gm/m2/ dose every 12 hours days 1-3), and consolidation followed by 3 years of monthly maintenance. (29) In the GIMEMA APL-2000 trial, the relapse rate for high-risk patients was lower when intermediate-dose cytarabine (1gm/m2/dose days 1-4) as part of the first cycle of consolidation, and 150 mg/m2 every 8 hours subcutaneously days 1-5 as part of the third cycle of consolidation) was given. (29% vs 2%, p=.0004). (30) If indeed the rate of relapse in extramedullary sites, particularly the CNS, is problematic in patients who relapse or perhaps among those patients presenting with an elevated WBC, intermediate-or high-dose cytarabine may be an important strategy to pursue.

The antibody M195, a mouse monoclonal antibody reactive with the antigen CD33 expressed on the cell surface can eliminate minimal residual promyelocytic leukemia cells in patients with relapsed APL after attaining a complete remission (CR) with ATRA. (31, 32) Gemtuzumab ozogamicin (GO), a humanized anti-CD33 monoclonal antibody chemically linked to the potent antibiotic calicheamicin, appears particularly active in patients with APL, perhaps attributable to the high expression of CD33 on leukemia promyelocytes. LoCoco and colleagues administered GO to 16 patients in molecular relapse. (33) Molecular CR was obtained in 91% of patients after two doses, and the remaining patients after the third dose. Further anecdotal reports confirm the apparent remarkable sensitivity in APL of GO. (34, 35) Although acute promyelocytic leukemia cells express CD33 strongly, they express relatively low levels of P-glycoprotein. GO has been shown to inhibit the growth of ATRA-resistant as well as ATO-resistant APL cells in a dose-dependent manner, (36) providing a rationale for the administration of combinations of all 3 agents, earlier in the natural history of the disease, perhaps in high-risk patients.

Arsenic Trioxide for Molecular Relapse of APL In a preliminary report, two patients in molecular relapse, identified at the time of routine screening while in first CR at 12 and 36 months, became PCR negative after one cycle of ATO given in standard-dose and schedule.(37) Both patients remain RT-PCR negative at 30 and 28 months without further therapy. One patient completed 4 cycles of ATO and the second was removed from study after 20 doses of the first cycle. Although the number of patients is very small, these results encourage further study of ATO in patients with molecular relapse to induce a second remission. The identification of the best subsequent treatment for patients achieving a second remission with ATO will require further clinical trials. Some post-remission therapy appears required for patients induced into second remission with ATO after definitive morphologic relapse. (38, 39) . Although autologous HSCT has become an attractive strategy for such patients, this kind of intensive approach may not be required when ATO is given in the setting of molecular relapse.

Given the remarkable activity of ATO in patients with relapsed or refractory disease, (38, 39) efforts have been undertaken to evaluate ATO in induction and consolidation. Investigation in Shanghai has combined ATO with ATRA in patients with newly diagnosed APL and report a faster time to CR and more profound reduction in minimal residual disease.(40) Estey and colleagues also have reported excellent results with combination therapy of ATO and ATRA. (41) ATO has also been combined with low-dose ATRA. (42) Remarkably, ATO as a single agent has excellent activity in patients with newly diagnosed APL. (43) (44) (45) In a study from Iran, 95 of 111 (86%) patients with newly diagnosed APL achieved CR. The DFS at 2 years was 64% (45) . The North American Intergroup has just completed a prospective randomized trial (C9710) that randomized patients to receive or not receive 2 cycles of ATO as a second course of consolidation following 2 cycles of daunorubicin and 1 week of ATRA. Patients continuing in CR are randomized to one of two maintenance schedules.

Preliminary laboratory studies suggest that other novel strategies may be effective in patients with APL. Approximately 30-35% of patients with APL have internal tandem duplications of the FLT3 gene. (19, 20, 46, 47) In general, data suggest the presence of FLT3 internal tandem duplication mutations confer less favorable prognosis. Internal tandem duplications of the FLT3 gene and PML/ RAR and the PML/RAR fusion transcript cooperate in the development of APL. (47) Small molecules, which inhibit mutations in the FLT3 gene, may be useful. All-trans retinoic acid induces CD52 expression in leukemic promyelocytes suggesting a potential role for alemtuzumab. (48) Increased angiogenesis, mediated by vascular endothelial growth factors (VEGF), but not basic fibroblast growth factor (BFGF), may play a role in the pathogenesis of APL (49, 50) . Earlier studies showed that VEGF is expressed in APL cells and that it is regulated by ATRA. (49) In vitro studies show that ATRA abrogates VEGF production by NB4 cells. Recent studies demonstrated that VEGF acts as a survival factor for leukemia promyelocytes and that the mechanism of action appeared to be independent of VEGF receptor. (50) These data suggest that pertubating internal VEGF may be a fruitful strategy to pursue. In a study from Iran, ATO was given to newly-diagnosed patients with APL and bone marrow vascular density was assessed by immunohistochemistry for van Willebrand factor and markers for CD31.(51) Bone marrow hot spot vascular density was reduced after treatment with ATO. Therefore, antiangiogenesis approaches may prove beneficial.

Long-term follow-up studies confirm excellent outcome and probable cure for the majority of patients with newly diagnosed APL. The major focus of therapy for patients with low-and intermediate-risk is now on reducing toxicity. However, patients with high-risk disease require new strategies. Arsenic trioxide is remarkably effective in patients with both the relapsed and refractory disease as well as in patients with previously untreated disease. HSCT has become an attractive strategy for patients in second CR when molecularly negative cells can be harvested. Such results provide the basis for considering these approaches for patients in first CR with high-risk disease. Novel strategies to pursue include monoclonal antibody therapy, FLT3 inhibitors, combinations of ATRA, ATO and GO, and antiangiogenesis approaches. 

The clinical utility of testing for the molecular heterogeneity of AML is now widely accepted. Chromosome abnormalities detected cytogenetically or using molecular techniques that identify fusion transcripts resulting from the cytogenetic abnormalities are required for disease diagnosis according to the current World Health Organization (WHO) classification of AML [21] . Defining molecular abnormalities at a minimum by cytogenetics, and, in the case of patients with normal karyotype, by molecular genetic techniques, is now considered mandatory for determining prognosis [28, [31] [32] [33] . Cytogenetic analysis of bone marrow is also recommended for documenting complete remission (CR) in patients with pretreatment abnormal karyotypes [12, 27, 33] . Moreover, reverse transcription-polymerase chain reaction (RT-PCR)-based techniques are required for documenting molecular remission and monitoring residual disease in acute promyelocytic leukemia [38] , and increasingly recommended in other types of AML [23] . Perhaps most importantly, since 1994, the genetic heterogeneity of AML has served as the basis of non-protocol post-remission therapy and is now increasingly used to select risk-adapted therapy [6, 7, 33] . Herein we will briefly review the prognostic significance of the molecular heterogeneity of AML with normal cytogenetics, focusing primarily on results from Cancer and Leukemia Group B (CALGB).

Among adults with de novo AML, 40 to 45% present with normal cytogenetics when rigorous criteria, requiring full analysis of at least 20 bone marrow metaphases, are used [10, 20] . In all CALGB studies, we strictly adhere to these criteria [10] . Unfortunately, many studies consider cases to be normal cytogenetically when fewer than 20 metaphases or only blood has been studied. Since it is possible for blood to be normal when marrow is abnormal, which was the case in approximately 5% of AML patients in the CALGB database for whom both marrow and blood had been studied [CD Bloomfield, personal communication, 2005] , this can lead to misclassification and should be discouraged.

CALGB data have shown that among cytogenetically normal adults with de novo AML under the age of 60 years, 82% achieve CR when treated with daunorubicin, cytarabine and etoposide with or without PSC-833, an inhibitor of the multidrug resistance gene product P-glycoprotein [16] . Among patients who achieve CR, 40 to 45% are cured when treated with 4 cycles of high-dose cytarabine (HDAC) or autologous peripheral blood stem cell transplant (SCT) in first CR [16] . year survival for patients with cytogenetically normal AML treated on CALGB 9621 [24] was 41%, the best outcome obtained to date on any CALGB trial. Obviously, it is important to determine which patients are currently cured and how the remaining 60% should be managed to increase their cure rate. Over the last 8 years, several studies have demonstrated that the outcome of younger adults with karyotypically normal de novo AML varies substantially depending upon the presence or absence of specific molecular genetic alterations. Below we will discuss the most extensively studied molecular markers in more detail.

The first molecular marker found to have major prognostic significance in AML with normal cytogenetics was the partial tandem duplication (PTD) of the MLL gene [11] . The MLL (ALL1, HRX, or HTRX) gene is the human homolog of the Drosophila trithorax gene, located at 11q23. MLL encodes an approximately 430kilodalton protein with histone methyltransferase activity that regulates the expression of a homeobox (HOX) group of genes involved in embryonic development and hematopoiesis.

The MLL PTD occurs in 8 to 11% of karyotypically normal adults with de novo AML, and usually involves exons 5 through 11 or, less frequently, exons 5 through 12 [11, 13] . Among patients with a normal karyotype, the MLL PTD is associated with significantly worse remission duration, although it is not associated with shorter overall survival. Indeed, in a number of subsequent studies, the MLL PTD has been found to be an independent adverse prognostic factor for remission duration [13, 18, 29] .

Obviously, a major goal of discovery of prognostic molecular markers is to assist in developing improved therapy for subsets of AML patients with normal cytogenetics. Recently, Whitman et al. [46] has suggested that the MLL wild-type allele is silenced in AML blasts harboring the MLL PTD, most likely as a result of epigenetic modifications. Because re-expression of the MLL wild-type allele is associated with selective sensitivity to cell death, these data suggest that therapy that includes DNA methyltransferase and/or histone deacetylase inhibitors should be explored in patients with MLL PTD.

The FLT3 gene, located at 13q12, encodes a class III receptor tyrosine kinase that has an important role in cell proliferation, differentiation and survival. Mutations of the FLT3 gene are among the most common genetic anomalies in AML patients with normal cytogenetics. These mutations include FLT3 internal tandem duplication (ITD), affecting exons 14 and 15, in up to 38% of patients, and activating point mutations of D835 within the activation loop in the tyrosine kinase domain (TKD, Asp835 mutation) in about 12-14% of cases. The FLT3 ITD and Asp835 mutations promote constitutive autophosphorylation of FLT3 protein, which, once activated, confers ligand independent proliferation [41] . Cytogenetically normal AML patients with FLT3 ITD have an inferior prognosis for both remission duration and survival [17, 22, 28] . In some analyses, the worst outcome has been conferred by FLT3 ITD coupled with the lack of a FLT3 wildtype allele, as first shown by Whitman et al. [45] , or a high FLT3 mutant/wild-type allele ratio [43] . In multivariable analyses FLT3 ITD and MLL PTD have both been independent predictors for remission duration [18] . FLT3 Asp835 mutations have not hitherto been correlated with inferior prognosis in cytogenetically normal patients, but because they are less frequent, larger clinical studies are necessary to determine their prognostic significance.

Overexpression of the wild-type FLT3 gene has also been reported in cytogenetically heterogeneous AML patients without FLT3 ITD or Asp835 mutations, and associated with inferior survival [36] . Preliminary results from a relatively small study suggest that higher FLT3 expression might also adversely impact on remission duration and survival of AML patients with a normal karyotype [25] . Larger studies are required to ascertain the prognostic impact of FLT3 gene overexpression in cytogenetically normal AML. Clinical trials using small molecule FLT3 inhibitors as targeted therapy are currently ongoing, following reports of evidence of their activity in refractory or relapsed AML patients [26, 42] .

The CEBPA gene, located at 19q13.1, encodes a member of the family of basic region leucine zipper (bZIP) transcription factors that is essential for granulopoiesis. Mutations in CEBPA occur in 15% of AML patients with normal cytogenetics [18] . Most CEBPA mutations have been found in AML FAB subtypes M1 or M2, suggesting the induction of a stage-specific block in the differentiation pathway [37] . Clinical studies have revealed that CEBPA mutations are associated with prolonged remission duration and overall survival in AML patients with normal cytogenetics [5, 18] . In multivariable analyses, CEBPA mutations have added information to that provided by MLL PTD and FLT3 ITD status with respect to remission duration and by FLT3 ITD status for survival [18] . Moreover, three patients with familial AML, two of whom had a normal karyotype, and a recently discovered deletion of a cytosine residue at nucleotide 212 of the CEBPA gene also had an excellent prognosis [40] .

The BAALC gene, mapped to 8q22.3, encodes a protein with no homology to any known proteins or functional domains. BAALC is expressed mainly in neuroectoderm-derived tissues and hematopoietic precursors, with no expression in mature marrow or blood mononuclear cells [1] . In the initial report, CALGB found that high BAALC expression in pretreatment blood adversely impacted remission duration and overall survival in karyotypically normal de novo AML patients under the age of 60 years who either lacked a FLT3 ITD (i.e., were FLT3wt/wt) or harbored FLT3 ITD but also expressed the wild-type FLT3 allele (i.e., were FLT3ITD/wt) [1] . In a recent larger study from the German DSIL group of adult AML with normal karyotype under the age of 60, high expression of BAALC mRNA in circulating blasts was an independent adverse prognostic factor for resistance to initial induction chemotherapy, remission duration and overall survival [3, 4] . On multivariable analysis for remission duration and overall survival, BAALC expression and FLT3 ITD both conferred prognostic significance. In another recent study, high BAALC expression predicted shorter disease-free and overall survival in karyotypically normal AML patients who did not harbor FLT3 ITD or CEBPA mutations [5] . Preliminary data suggest that allogeneic SCT in first CR might overcome the adverse prognostic effect of high BAALC expression [4] .

The NPM1 gene, located at 5q35, encodes nucleophosmin, a multifunctional protein with a chaperone function and a prominent nucleolar localization that shuttles between the nucleolus and cytoplasm. In addition to preventing nucleolar protein aggregation, nucleophosmin has been implicated in the regulation of ribosomal protein assembly and their nucleocytoplasmic transport, as well as the regulation of the ARF and p53 tumor-suppressor pathways [15] .

Mutations of exon 12 of the NPM1 gene result in aberrant cytoplasmic localization of the nucleophosmin protein, and occur predominantly in de novo AML patients with a normal karyotype [15] . In fact, NPM1 mutations represent hitherto the most common genetic rearrangement in cytogenetically normal AML, being reported in 47 to 62% of patients with a normal karyotype [8, 14, 15, 39] . Furthermore, it has been shown that patients with mutated NPM1 carry FLT ITD approximately twice as frequently as those with wild-type NPM1 alleles [14, 15, 39] .

The prognostic significance of mutations in the NPM1 gene has been demonstrated by several [14, 15, 39] , but not all [8] , recent studies. Falini et al. [15] reported that cytoplasmic expression of nucleophosmin, highly correlated with the presence of NPM1 mutation, was an independent favorable prognostic factor for achievement of CR. A subsequent study has demonstrated both a significantly better CR rate and longer event-free survival in patients with NPM1 mutations as opposed to those without, but the favorable prognostic impact of NPM1 mutations was not observed in patients who also harbored a FLT3 ITD [39] . Likewise, Döhner et al. [14] identified a significant interaction between NPM1 and FLT3 ITD, where patients with mutated NPM1 had a higher CR rate and overall survival only in the absence of FLT3 ITD. In multivariable analyses, the combined status of mutated NPM1 and wild-type FLT3 was shown to constitute an independent favorable prognostic factor for remission duration and survival [14] .

The ETS-related gene, ERG, located at chromosome band 21q22, and other members of the ETS family are downstream effectors of mitogenic signaling transduction pathways and are involved in key steps regulating cell proliferation, differentiation, and apoptosis [34, 35] . We have recently reported that ERG is frequently overexpressed in AML patients with prognostically unfavorable complex karyotypes that contained cryptic amplification of chromosome 21 [2] . Because ERG overexpression was not always associated with genomic amplification and was also detected in cytogenetically normal AML [2] , we hypothesized that overexpression of ERG might confer an aggressive malignant phenotype in a subset of AML patients with a normal karyotype and potentially impact on their prognosis. Indeed, we found that high ERG expression in pretreatment blood adversely affected remission duration and overall survival in de novo AML patients with normal karyotype under the age of 60 years treated on CALGB 9621 [29] . In multivariable models, high ERG expression and FLT3 ITD independently predicted worse remission duration and overall survival. When the analysis was restricted to the more favorable subset of patients lacking FLT3 ITD or expressing both FLT3 ITD and the wild-type allele, high ERG expression and MLL PTD both impacted on remission duration. With regard to overall survival, we found an interaction between expression of ERG and BAALC, with ERG overexpression predicting shorter survival only in low BAALC expressers [29] . These findings await corroboration.

Gene-expression profiling using DNA microarray technology is a powerful tool allowing analysis of expression of thousands of genes in one experiment. Early studies demonstrated that it is possible to distinguish AML from ALL correctly based on gene-expression profiles [19] . Successive studies have revealed that several cytogenetically and molecularly defined AML subtypes such as t(15;17)/ PML-RARA, t(8;21)/AML1(RUNX1)-ETO(CBFA2T1) and inv(16)/t(16;16)/CBFB-MYH11 display characteristic gene-expression signatures [9, 44] that, not surprisingly, correlate with clinical outcome [44] . Moreover, novel gene clusters, seemingly not corresponding to cytogenetic aberrations, have also been identified; some have had prognostic significance [44] .

However, normal karyotype AML seems to have more heterogeneous geneexpression profiles; patients with these karyotypes have been found in more than one cluster based on dissimilar patterns of gene expression. In a study by Bullinger et al. [9] , a relatively small cohort of karyotypically normal patients segregated mainly into two clusters with significantly different survival. This promising finding suggested that gene-expression profiling could potentially become useful in predicting clinical outcome, but it required verification, especially because specimens from AML patients with normal cytogenetics in another study [44] segregated into several, instead of two, distinct clusters. CALGB has recently confirmed the prognostic significance of the gene-expression signature identified by Bullinger et al. [9] using the Affymetrix U133 plus 2.0 GeneChips (Affymetrix, Santa Clara, CA) [30] . More studies are necessary to assess the role of microarray gene-expression profiling in clinical practice and prognostication of AML patients with a normal karyotype.

Recent studies have shown that karyotypically normal AML is molecularly heterogeneous. Many molecular genetic alterations have been shown to influence the clinical outcome of cytogenetically normal AML patients [1, 4, 5, 9, 11, 13-15, 17, 18, 22, 28-30, 39, 45] , although the prognostic significance of some genetic markers have been demonstrated only in single studies, e.g., ERG overexpression [29] , or in studies comprising a relatively small number of patients, e.g., Bullinger et al.'s gene-expression signature [9, 30] . Moreover, relatively few studies have analyzed multiple genetic markers simultaneously, making assessment of the relative contribution of individual markers in predicting clinical outcome difficult. Thus, there is an urgent need to conduct clinical trials that would test all known molecular markers and perform microarray expression profiling. It is hoped that such trials will distinguish subsets of patients with normal AML karyotype and different clinical outcome as well as lead to the development of more effective targeted therapies for specific patients. Recognizing the importance of these new tests, the U.S. National Institutes of Health have funded a web site to help locate laboratories that perform various genetic assays. This website can be accessed at http://www.genetests.org/ servlet/access?id=8888892&key=6syrDpoKyLinE&gry=INSERTGRY&fcn=y&fw= yW82&filename=/.

Introduction A period of rapid improvement of treatment outcome of adult acute lymphoblastic leukemia (ALL) in the last decades induced by intensive chemotherapy, increased use of stem cell transplantation, improved supportive care and more experience of clinical centers was followed more recently by a period of stagnation. Further improvement seemed to be possible only in distinct subtypes of ALL. Thus it is fortunate that in the last few years promising new treatment modalities came up 1 2. The most promising approaches are probably targeted therapies with mechanisms of action different from conventional chemotherapy. They include molecular targeting with kinase inhibitors such as Imatinib but also antibody therapy directed to surface antigens of leukemic blasts (reviews in 3 4).

ALL blast cells express a variety of lineage specific antigens and combinations of antigens which are used for establishment of diagnosis and definition of immunologic subtypes.. Therapy with monoclonal antibodies (MoAb) directed to these antigens is an alternative attractive treatment approach in ALL since it is targeted, subtype specific and compared to chemotherapy has different mechanisms of action and side effects. Antibody therapy may therefore be offered particularly to patients in whom intensification of chemotherapy is impossible and it may act on leukemic clones which are resistant to cytostatic drugs. Also synergistic effects of antibody therapy and chemotherapy can be utilised.

There is some evidence that the activity of antibodies depends on the degree of antigen expression on the cell surface. A prerequisite for MoAb therapy was generally the presence of the target antigen on at least 20% of the leukemic blasts. With higher incidence of antigen positive cells the chance of clinical response probably increases further. Also the antigen expression on individual cells is of interest although few data are available so far.

Rituximab is a chimeric human/mouse monoclonal antibody to CD20, a surface antigen which is expressed on normal and malignant B-lymphocytes. It is however not expressed on normal stem cells. CD20 has a functional role in Bcell growth. For Rituximab various mechanisms of action have been identified by in vitro studies including complement-dependent cytotoxicity, antibodydependent cellular cytotoxicity and induction of apoptosis 5. The addition of Rituximab to classical CHOP-based regimens led to a significant improvement of outcome in diffuse large B-cell lymphoma 6 without additional significant toxicity. New applications are currently tested in NHL e.g. as maintenance, use in indolent lymphoma or in the peritransplant setting show additional modalities (review in 7). CD20, defined as expression on more than 20% of the blast cells is however also present on one third of B-precursor ALL blasts, particularly in the elderly patients (40-50%), and the majority of mature B-ALL blast cells (80-90%) (data from the GMALL Central Lab for Immunophenotyping, S.Schwartz, E.Thiel, Berlin). This provides a rationale to explore Rituximab in B-precursor ALL, mature B-ALL and Burkitt's lymphoma.

Rituximab in mature B-ALL and Burkitt's NHL The outcome of mature B-ALL and Burkitt's NHL has been improved substantially in the past decades by treatment according to short intensive protocols derived from pediatric studies 8. These protocols are mainly based on intensive cycles with high-dose methotrexate and fractionated cyclophosphamide or ifosfamide. In adult patients however the options for treatment intensification by increased dose-intensity e.g. of methotrexate are limited. Therefore -based on the favourable results in other high-grade B-NHLthe German Multicenter Study Group for Adult ALL (GMALL) initiated a study with Rituximab in combination with chemotherapy (schematic schedule see figure 1 ). The regimen with six 5-day chemotherapy cycles is based on previous GMALL studies 8. Major new features are (1) 8 applications of Rituximab (before each chemotherapy cycle and 2 applications for consolidation) and (2) a new cycle with high-dose cytarabine beside other drugs (cycle C). Patients younger than 55 years receive 6 cycles (ABCABC) with 1.5 g/m methotrexate and older patients a dose reduced regimen (methotrexate 0.5 g/m) without cycle C (ABABAB). In stage I/II without extranodal or mediastinal involvement treatment ends after 4 cycles. Radiation after 6 cycles is foreseen in patients with residual tumor, mediastinal tumor or CNS involvement at diagnosis. Between 9/02 and 6/03 82 patients (>=15 yrs) from 39 centers entered the new protocol and 53 were evaluable. CR (including 1 CRu) after 2 cycles (AB) was achieved in 10/11 B-ALL pts (91%). In 26 patients with Burkitt-NHL the response rate (15 CR/ 10 PR) after two cycles was 96% 9. Rituximab was administered without excess toxicity. In the meantime more than 170 patients have been included and the results are confirmed. The overall survival is around 80% for Burkitt-NHL and 70% for mature B-ALL. Elderly patients above 55 years with these diseases had a poor outcome with the former protocol B-NHL90 without rituximab 10. The CR-rate in 45 patients was 71% and the overall survival 39%. In the new protocol including Rituximab in 26 evaluable patients the CR-rate increased to 81% and the overall survival at 1.5 years was 84% (p=.03 compared to study B-NHL90) 11.

A similar approach was investigated at the MD Anderson Cancer Center. Scheduling was slightly different with this regimen since Rituximab was added to the Hyper-CVAD regimen in the beginning and the end of each cycle. The total number of doses was 8 as well. In 31 patients with newly diagnosed Burkitt's NHL or mature B-ALL 86% complete responses were observed and the 3 year survival was 89%. The authors observed a significant reduction of relapse rate and an improvement of outcome particularly in elderly patients. Also in this study there was apparently no additional toxicity compared to the previous protocol with chemotherapy only 12.

Rituximab for elderly patients with CD20 positive B-precursor ALL Despite the major peak of ALL in childhood the incidence also increases in the elderly beyond 50 years. Outcome of these elderly ALL patients is very poor. In a survey including 12 studies with 370 pts (50-88 yrs) the mean CR rate was 50% and the rate of continuous CR (CCR) 13%. Also in a first prospective pilot study of the German Multicenter Study Group for Adult ALL (GMALL) for elderly ALL pts > 65 yrs with a moderate intensive chemotherapy regimen the remission rate was 46% and the survival after 2 years was 15% 13.

Approximately 50% of the patients with B-precursor ALL (common-or pre-B-ALL) are CD20 positive at an expression level above 20%. In elderly ALL patients where options for chemotherapy intensification is limited the addition of a second treatment approach is particularly important. In a current GMALL study CD20 positive patients receive Rituximab before each cycle of the above mentioned dose-reduced chemotherapy for a total of 8 applications. For an interim analysis 26 patients were evaluable. The CR rate in CD20 positive patients was 63% and the overall survival after 1 year was 54% 11. Thus in Bprecursor ALL the combination of chemotherapy and Rituximab is feasible but long-term results are awaited. The risk of infections during induction therapy and in remission remains however a major problem although there are no hints that this problem is aggravated by Rituximab therapy.

Rituximab for Younger Patients with CD20 Positive B-Precursor ALL Based on the experience in older patients the GMALL has also added Rituximab to protocols for younger patients with CD20 positive B-precursor ALL. In the current GMALL protocol 07/2003 patients with B-precursor ALL are subdivided into three risk groups. Very high risk for Ph/bcr-abl positive ALL, high risk for patients with WBC above 30.000/l or late achievement of CR and standard risk for patients without one of these risk factors. After introduction of Imatinib for very high risk patients, high-risk patients with B-precursor ALL are the most unfavourable subgroup of adult ALL. By addition of rituximab before induction phase I, II and first consolidation a reduction of tumor load before the scheduled stem cell transplantation is attempted.

In standard-risk ALL patients with B-precursor ALL 8 applications of Rituximab before induction and consolidation cycles are given in order to improve remission quality. The success of this approach is evaluated by quantiative measurement of minimal residual disease.

The current treatment strategy of the MD Anderson Hospital also includes Rituximab combined with the Hyper-CVAD regimen in the treatment of Bprecursor ALL together with other modifications. Interim results showed a favourable disease free survival, however some cases of death in CR in elderly patients with CD20 positive ALL 14.

Whereas results of these studies are awaited, several case reports supported the hypothesis that Rituximab is capable to induce molecular remissions in the state of minimal residual disease (MRD) in B-precursor ALL 15 16. In pediatric ALL treatment with weekly application of rituximab even in overt relapse of B-NHL or B-ALL led to responses in individual cases 17 18.

Overall antibody therapy with Rituximab is a promising new approach for the treatment of adult ALL which deserves systematic studies in larger patient cohorts. The fact that single responses with Rituximab monotherapy were even achieved in advanced ALL demonstrates that the approach is in principle active in ALL. These findings are strongly supported by the very favourable results in mature B-ALL and Burkitt's NHL. The efficacy in B-precursor ALL still has to be proven. With respect to indications it seems already clear that monotherapy with A1 B1 P C1 A2 B2 C2 antiCD20 antiCD20 antiCD20 antiCD20 antiCD20 antiCD20 1 Patients 15-55 years antiCD20 antiCD20 1 2 5 8 12 15 18 Weeks 21 24 28 A1 * B1 * P A2 * B2 * A3 * B3 * antiCD20 antiCD20 antiCD20 antiCD20 antiCD20 antiCD20 1 antiCD20 antiCD20

Patients > 55 yrs MoAbs in full clinical relapse (or in de novo ALL) leads to a CR in rare cases only. Efficacy can be increased by combination with chemotherapy -parallel or sequential. The optimal schedule remains to be defined. Furthermore it is of particular interest to evaluate antibody therapy in low level disease with the aim to achieve molecular remissions. This applies for the status after chemotherapy as well as for SCT. In this setting the question of response evaluation comes up. It remains to be demonstrated whether highly sensitive methods for MRD analysis will be applicable for this purpose.

Leukemias have traditionally served as model systems for research on neoplasia because of the easy availability of cell material from blood and marrow for diagnosis, monitoring and studies on pathophysiology. Beyond these more technical aspects, chronic myeloid leukemia (CML) became the first neoplasia in which the elucidation of the genotype led to a rationally designed therapy of the phenotype. Targeting of the pathogenetically relevant BCR-ABL tyrosine kinase with the kinase inhibitor imatinib has induced remissions with almost complete disappearance of any signs and symptoms of CML. This therapeutic success has triggered an intensive search for target structures in other cancers and has led to the development of numerous inhibitors of potential targets which are being studied in preclinical and clinical trials worldwide.

Model Disease CML CML has served as a model for other cancers due to its multistep evolution with several defined stages (chronic phase, acceleration, blast crisis), its regular association with a defined cytogenetic translocation t(9;22)(q34;11), the elucidation of molecular pathogenesis and the successful development of a molecular targeted therapy. The term leukemia was first coined by Virchow in 1845 when he realized the neoplastic nature of purulunt matter ("weißes Blut") in patients with what later was designated CML. The detection of the Philadelphia chromosome (Ph) in 1960 provided a marker that almost unequivocally defined the disease. In 1973, it was recognized by Janet Rowley that the basis of the Philadelphia chromosome was a reciprocal translocation between the long arms of chromosome 9 and 22. The molecular structure of this translocation was clarified in the early 1980s, and in 1990 it was shown by Daley and colleagues that BCR-ABL transformed cells in vitro and that BCR-ABL transfected marrow cells induced leukemia in mice. This demonstration of pathogenetic relevance of the BCR-ABL fusion gene then led to the search for inhibitors and to the introduction of the tyrosine kinase inhibitor imatinib into clinical investigation and application in 1998. The introduction of a rationally designed pharmacotherapy directed against a pathogenetically relevant target marks the preliminary end point of 140 years of attempts to treat and cure CML (1).

The first drug that was reported active in CML was arsenic in 1865. Currently, arsenic is reintroduced into CML management as second line treatment for combination with imatinib (2) . Therapy was palliative during the first century of CML treatment which included splenic irradiation, various cytostatic agents of which busulfan was standard for almost three decades and intensive combination therapy. The intention of the treatment became curative with the introduction of stem cell transplantation in the 1970s (1). A prolongation of survival could be offered by interferon in combination with hydroxyurea or low dose ara-C, particularly in low risk patients and in patients who achieve a cytogenetic remission (3). The introduction of imatinib into CML therapy marks a major advance in CML treatment with regard to efficacy and lack of adverse reactions. Its mechanism of action is blocking the ATP-binding site of the BCR-ABL tyrosine kinase with high affinity and high specifity. After imatinib had been shown to inhibit BCR-ABL-positive cell lines in vitro (4), phase I trials started in 1998 and phase II trials in 1999. Imatinib showed good efficacy and tolerability in patients who had failed IFN treatment. The beneficial effect of imatinib was demonstrated in chronic phase, advanced phase and in blast crisis as well as in Ph+ acute lymphatic leukemia (ALL, 5).

In a phase III trial on 1106 non-pretreated patients in early phase CML randomized between imatinib and IFN in combination with low dose ara C, the imatinib group achieved complete hematologic remissions in 98%, partial cytogenetic remissions in 92% and complete cytogenetic remissions in 86% of cases (54 month data, update 2004) (6). The time to complete hematologic remission was much shorter with imatinib (about 90% after 3 months) than with IFN. Similar to the effects observed with IFN, the achievement of complete cytogenetic remissions is followed in most patients by a continuous decline of BCR-ABL transcript levels. Major parameters with prognostic impact were any cytogenetic response after six months and major cytogenetic response after 12 months of therapy.

On the basis of survival data of IFN-treated CML-patients who achieved complete cytogenetic remissions a 10-year-survival rate of at least 51% was estimated for imatinib-treated patients (7) . It can be concluded that imatinib is superior to IFN with regard to response rate, progression free survival and adverse effects. No definite data exist yet as to long-term survival and late toxicity although prolongation of survival by imatinib is expected.

Molecular monitoring of BCR-ABL transcript levels with quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) technology in patients who have achieved a complete cytogenetic remission has become an important asset of long term CML management. Real time quantitative RT-PCR using specific fluorescent hybridization probes and standard procedures with internal controls allow a rapid and accurate analysis (8) (9) (10) .

Early reduction of BCR-ABL transcript levels predicts cytogenetic response and favourable clinical course in imatinib-treated chronic phase CML patients. Low levels of residual disease have been associated with continuous remission. The degree of molecular response correlates directly with progression-free survival (9,10). The persistence of BCR-ABL transcripts even after prolonged imatinib treatment in most patients argues against a prospect of cure by imatinib alone and for additional therapeutic measures. Similar observations have been made earlier after allografting. Transplanted patients with complete disappearance of BCR-ABL transcripts within 6-12 months have been found to have excellent prospects for a successful transplantation outcome and probably cure. Transplanted patients with persistence of BCR-ABL transcripts, or reappearance of transcripts after initial disappearance have an increased risk of relapse.

Detection of minimal residual disease (MRD) is now becoming routinely implemented in protocols for guiding therapy and for evaluation of new treatment modalities. The lack of standardization of the methodology represents a major barrier in the comparison of data generated in different studies. Therapeutic response can be expressed in three ways: (i) Calculation of the ratio of mRNA transcripts of target to reference gene, e.g. ratio BCR-ABL/ ABL, (ii) Individual calculation of the relative molecular response: i.e. comparison of the MRD level after therapy vs pretherapeutic level, and (iii) use of a lab-specific reference point, e.g. a pool of diagnostic samples for calculation of the log reduction. In the IRIS trial, a log reduction >3 after 12 months of imatinib therapy was accompanied by a 95% relapse free survival after 54 months and defined as "major molecular response" (MMR, 10, update 2005). The reference sample, however, is not available for widespread distribution. There is a relationship between the log reduction approach and the ratio of BCR-ABL to total ABL transcripts. Using standardized methods for the quantification of BCR-ABL and ABL transcript with an identical plasmid dilution for both transcript types (9), a 3-log reduction is actually identical to a ratio BCR-ABL/ABL=0.12%.

Several open questions remain, notably those concerning the development of imatinib resistance which is rare in early chronic phase, but increases in frequency along the course of the disease (11) . Essentially two mechanisms underlie the development of imatinib resistance: 1. Mutations of the ATP-binding site of the BCR-ABL tyrosine kinase and 2. clonal evolution with aberrant karyotypes ultimately leading to blast crisis. Pharmacologic mechanisms including activation of multi drug resistance proteins may cause variations in the individual intracellular imatinib concentrations which may contribute to the development of resistance.

Detailed sequence analysis has been performed to elucidate which mutations are responsible for the development of imatinib resistance. More than 30 different mutations have been recognized which are detailed elsewhere (11) (12) (13) . The prognostically most serious mutations concern the so-called "Ploop" domain of the tyrosine kinase. P-loop mutations have been associated with an especially poor prognosis (13) , but cessation of imatinib therapy and alternative therapy with other drugs seem to be able to improve prognosis. Novel methods are available to screen for small clones of mutated leukemic cells, e.g. D-HPLC. The impact of the results of such assays needs to be explored in prospective clinical trials. Several approaches appear feasible to prevent or overcome imatinib resistance: 1. to increase imatinib dosage to 600 or 800 mg, 2. to combine imatinib with other drugs of known anti-CML-activity, or 3. more efficient alternative BCR-ABL inhibitors. The increase of imatinib dosage has been previously shown to improve response in patients with accelerated disease. Accelerated disease was found to have higher response rates with 600 mg imatinib than with 400 mg. Kantarjian et al. have reported in a historical comparison that higher cytogenetic and molecular remission rates can be achieved in shorter time intervals with an imatinib dosage of 800 mg daily as compared to 400 mg in chronic phase CML (14) . The disadvantage of the higher imatinib dose is a higher rate of adverse effects, in particular myelosuppression and fluid retention. It is unknown whether the effect of high dose imatinib is sustained and provides a survival benefit.

Combinations of imatinib with other drugs have been extensively analyzed in vitro and have shown that a number of drugs are synergistic with imatinib in vitro . Of particular interest were the combinations of imatinib with IFN or low dose ara-C. The feasibility of the combinations of imatinib with IFN (Pegasys, Peg-Intron) and low dose ara-C has been shown in phase I and II studies (15) .

On the basis of these studies, randomized trials were designed by national study groups in Germany, France, the United Kingdom, and USA to compare imatinib monotherapy at 400 mg with imatinib in various combinations (IFN, ara-C) and dosages (600 mg, 800 mg). The German CML Study IV, started recruitment in July 2002. By December 2005, 700 patients had been randomized. The study compares imatinib monotherapy at 400 mg vs the combination of imatinib plus IFN vs imatinib plus low dose ara-C vs imatinib after IFN failure (16) . The sequential treatment concept of imatinib after IFN failure is supported by the modes of action of the two drugs. IFN has been shown to induce a T-cell response against proteinase 3 which is associated with complete cytogenetic remission. No such response has been observed with imatinib which may even inhibit T-cell activation (17) . It therefore might well be that the sequential as compared to the simultaneous treatment approach provides an advantage. After imatinib failure allogeneic transplantation is recommended for all patients who have a donor and are eligible for the procedure. In patients above age 45, the feasibility and efficacy of reduced intensity conditioning will be analyzed in a randomized fashion. This goal will hopefully contribute to a better cost effectivity.

The emergence of resistance has led to a search for downstream targets of the BCR-ABL kinase that may mediate the altered growth properties of BCR-ABL-transformed cells. Identification of signaling pathways downstream of ABL tyrosine kinase may increase our understanding of the pathogenesis of CML and suggest strategies to improve clinical treatment of the disease. Farnesyltransferase inhibitors enhance the antiproliferative effects of imatinib against BCR-ABL-expressing cells, including imatinib-resistant cells. Early clinical studies using a combination of imatinib and farnesyltransferase inhibitors in advanced phase CML patients demonstrated feasibility but showed only moderate activity, probably due to clonal evolution with novel molecular or cytogenetic aberrations in addition to BCR-ABL not responding to farnesyltransferase inhibitors.

The serine/threonine protein kinase mTOR (mammalian Target of Rapamycin) is a downstream component of the PI3-Kinase/Akt pathway, and plays an important role in controlling cell growth and proliferation. The mTOR pathway is constitutively activated by BCR-ABL in CML cells. Two of its known substrates, ribosomal protein S6 and 4E-BP1, are constitutively phosphorylated in a BCR-ABL-dependent manner in BCR-ABL-expressing cell lines and CML cell lines. These data suggest that BCR-ABL may regulate translation of critical targets in CML cells via mTOR.

The effect of rapamycin in three different imatinib-resistant BCR-ABL mutant cell lines (Ba/F-BCR-ABL T315I, G250E, M351T) has been described. Rapamycin alone showed inhibition of proliferation to a degree that would be predicted were mTOR a critical downstream effector of BCR-ABL, while the combination of low-dose rapamycin with imatinib markedly enhanced this growth inhibitory effect. The synergy between rapamycin and imatinib, occurring at doses well below typical serum levels obtained during monotherapy with each of these agents represents a strong argument in favor of investigating the clinical activity of the combination.

A third approach to overcome imatinib response is the development of new more efficient BCR-ABL tyrosine kinase inhibitors. Several new compounds have been reported, two of which have entered clinical trials (AMN107 and dasatinib, [18] [19] . Target structures of AMN107 are BCR-ABL, c-KIT and PDGFR, of dasatinib BCR-ABL, c-KIT, PDGFR and SRC. For ABL inhibition, AMN107 and dasatinib are more potent than imatinib and have been shown to retain activity against most, but not all, imatinib-resistant BCR-ABL mutants. Phase I and II data for both drugs are promising and patients being resistant or intolerant to imatinib should be enrolled into current trials.

Although allografting is still considered to be the only potentially curative approach to CML, transplantation numbers have dropped significantly in the imatinib era due to transplantation associated mortality and morbidity. A trend towards lower mortality rates after related donor transplantations has been noted, but overall mortality seems to stay unchanged due to the higher proportion of unrelated donor transplantations and an increased age of transplanted patients. Categorization of patients according to transplantation therefore becomes increasingly important.

The EBMT score by Gratwohl et al. (20) allows the recognition of patients with especially low or high transplantation risks. Current management of newly diagnosed CML patients therefore has to include the evaluation of patients according to risk profile and transplantation risk.

The realization that donor lymphocytes constitute the most relevant factor in eliminating residual disease in CML has led to a reduction of intensity of conditioning regimens prior to allografting. Low intensity conditioning permits allografting also in older patients and patients with other thus far disqualifying conditions. Currently this new approach is studied for long-term efficacy and cost effectiveness in many centers as described above for CML Study IV.

The advent of selective tyrosine kinase inhibitors has significantly changed CML therapy. However, despite promiding results patients should be identified in whom treatment requires optimization, either by dose escalation of imatinib or combination with other drugs. In case of resistance, novel tyrosine kinase inhibitors are available within clinical trials. In addition to hematologic and cytogenetic monitoring, molecular surveillance of response and resistance is essential for therapeutic decisions.

Clinical Development of Gemtuzumab Ozogamicin (Mylotarg) in Patients with CD33-Positive Acute Myeloid Leukemia R.A. LARSON University of Chicago, Chicago IL, USA

Three multicenter, open-label, single-arm phase II studies were conducted to evaluate the efficacy and safety of gemtuzumab ozogamicin (GO; Mylotarg), an antibody-targeted chemotherapy for CD33-positive acute myeloid leukemia (AML).

Patients with CD33+ AML in untreated first recurrence were enrolled. Patients with prior myelodysplasia (MDS) were excluded. Patients received monotherapy with GO 9 mg/m2 as a 2-hour intravenous infusion in 2 doses separated by 2 weeks. Patients were evaluated for remission, survival, and treatment-emergent adverse events.

The regulated expression of differentiation-associated genes during normal hematopoiesis is governed by the epigenetic process of cordinate gene activation by promoter demethylation (Egger et al. 2004 ), e. g. during the activation of the myeloperoxidase (MPO) and lysozyme genes during myeloid differentiation (Lübbert et al. 1996) , and gene silencing of fetal globin genes (-globin) during the switch to adult erythropoiesis (Saunthararajah and DeSimone 2005) . Therefore DNA methylation has provided a target for inducing differentiation by pharmacological inhibition of this process to achieve cellular differentiation (Pinto et al. 1984 ). More recently, the aberrant hypermethylation of many growth-regulatory and several proapoptotic genes has led to further acknowledgement of hypermethylation as a molecular target for novel treatment approaches in hematologic neoplasias, hemoglobinopathies and solid tumors (Lü bbert et al. 2000) . The discovery of the DNA methylation inhibition by analogues of cytidine, i. e. the azanucleosides 5-azacytidine (Vidaza) and 5aza-2´-deoxycytidine (Decitabine, Dacogen) was made by Jones and Taylor (1980) . This seminal discovery was based on the induction of terminal cellular differentiation to functional myocytes chondrocytes and adipocytes in an embryonal fibroblast model. Since then, the development of both azanucleosides, and more recently novel small molecules also aimed at DNA inhibition, has followed a slow, arduous and often rocky path (Lyko and Brown 2005) . With the more recent establishment of epigenetic silencing as a major step during carcinogenesis, development of these treatment approaches has been spurred, now resulting in clinical trials which combine these epigenetically active agents with other compounds not primarily acting as cytostatic drugs but as "biological response modifiers". We will summarize the development of demethylating therapy from the initial use of these agents as mere cytostatic drugs, used at high doses similar to cytarabine, to their present use at low doses, strongly favouring demethylation activity over cytotoxic activity, with schedules allowing outpatient and non-intensive treatment even in older patients with myeloid neoplasias who so far have been a major therapeutic challenge.

The Past: Azanucleoside Use at Intensive Schedules in Leukemia and Solid Tumors Shortly after the synthesis of the azanucleoside drugs 5-azacytidine and decitabine, both drugs entered the clinical arena, with development for leukemia treatment in North America (5-azacytidine, somewhat later decitabine) and numerous phase I/II trials in various solid tumors in North America (5azacytidine), Europe and South America (decitabine). The overall experience with 5-azacytidine used in leukemia clinical trials at doses that were equitoxic to standard-dose to high-dose cytarabine, and almost always used in combination with other antileukemic agents, was not inferior to cytarabine but burdened with marked toxicity, particular leukopenia, nausea/vomiting and liver and kidney toxicity (Glover 1987) . A somewhat similar experience in acute leukemias was made with decitabine, which proved to be equivalent to high-dose cytarabine when used in conjunction with anthracyclines both in relapsed/refractory AML (Willemze et al. 1997 ) and, in single a study, in de novo AML (Schwartzmann et al. 1997) . However, at the high doses used between the 1970s and early 1990s, repetitive courses of this treatment were difficult to apply and the toxicity spectrum at these doses proved prohibitive for use in older patients with leukemia.

The experience with both drugs in solid tumor treatment was overall disappointing, with low rates of objective responses (Lü bbert 2000), which in retrospect was most likely due to schedules not allowing for continuous exposure over repetitive courses to this drug. Therefore, this development was not competing with other compounds more effective in solid tumors at the time.

The Present: Low-Dose Azanucleosides as Effective Continuous Treatment of Myelodysplasia The pioneering work of introducing low-dose treatment with azanucleosides in preleukemia, acute myeloid leukemia and hemoglobinopathies started with the seminal discovery of effective hemoglobin F induction in a patient with + thalassemia (Ley et al. 1981 ). These results provided first in vivo proof of "transcriptional therapy" by reactivation over normally silent, hypermethylatedglobin gene locus. Pinto and colleagues (Aviano, Italy) built on this discovery by first using low-dose infusional schedules of decitabine in elderly and frail patients with AML and high-risk MDS (Pinto et al. 1989 ). The rationale for using non-cytotoxic doses of this drug was also based on induction of differentiation via demethylation of master genes of myeloid maturation.

The development of 5-azacytidine in the USA led to the pivotal phase III study by the CALGB (coordinated by Prof. L. R. Silverman) demonstrating that a treatment with subcutaneous 5-azacytidine given on 7 consecutive days over at least 4 months results in prolonged time to AML in treated patients compared to those receiving best supportive care only (Silverman et al. 2002) . A confirmatory trial comparing this treatment to either best supportive care alone, low-dose cytarabine or induction chemotherapy is ongoing. Other development of this type of treatment is aimed at comparing schedules that do not involve 7 days treatment but 5 days or 12 treatment (Lyons et al. 2005) .

In Europe, studies by Pinto and Wijermans have shown an overall response rate of ca. 50% to repeated courses of 3-day infusions of low-dose decitabine, with ca. 25% complete partial remissions and 31% cytogenetic responses (Wijermans et al. 2005 ). These encouraging data let to phase III studies in Europe and North America, one of which has been completed (Saba et al. 2005), whereas the EORTC/German MDS Study Group trial is ongoing. This trial targets patients with high-risk MDS, with poor-risk cytogenetics as the unifying inclusion criterion, since patients with these normally very poorly responding karyotypic abnormalities respond particularly well to decitabine (Wijermans 2005) , Fig. 1 . This ongoing trial aims at application of a maximum of 8 treatment courses (equalling 1 year of treatment) since a lower than expected rate of complete and partial remissions in the North American phase III decitabine trial is most likely due to many patients being taken of study before receiving more than 3 courses (Saba et al. 2005). However, it is well established for both azanucleosides when given at low doses that continuous treatment is necessary to achieve the full effect, and late responses occurring after more than 6 months are not at all infrequent with these drugs.

Only one study has addressed the question of activity of 5-azacytidine at the low-dose schedules with doses established for MDS treatment (Lee et al. 1990 ). This has been examined in (mostly pretreated) AML patients (by FAB classification), without a clear effect of this approach. In contrast, decitabine at the dose and schedule effective in MDS is showing activity in an ongoing trial of older AML patients ineligible for induction chemotherapy who receive 4 courses of 3 day infusions, followed in responding patients by outpatient maintenance with 3 1-hour infusions repeated every 6-8 weeks until relapse or progression (Lü bbert et al. 2005), Fig. 2 .

The rationale for using DNA demethylating activity in patients with hematologic neoplasia (and in the future in solid tumors) is based on the discovery of multiple hypermethylated genes in the malignant cells. Proof of principle for in vivo demethylation has been given for the p15/INK4b gene which is hypermethylated in MDS and leukemia, and demethylated with decitabine therapy (Daskalakis et al. 2002; Issa et al. 2004; Gore et al. 2004) . Future studies will be directed at other genes which may also become reactivated via demethylation in vivo. However, since with both available demethylating agents almost half the patients do not show a response, combination treatments with other drugs are clearly warranted. The most promising combination is with an inhibitor of histone deacetylase (HDAC), since an in vitro synergism has been described (Cameron et al. 1999). Apart from several new drugs in development, valproic acid has been demonstrated as a very active HDAC inhibitor, and also has single-agent activity in low risk-MDS (Kü ndgen et al. 2005). Other combination partners include all trans retinoic acid or chemotherapeutic agents such as carboplatinum. It is to be expected that with these developments, epigenetic therapy will be more broadly used particularly in those patients who not tolerate aggressive chemotherapy i. e. older and/or patients with comorbid conditions.

Over the past four to five decades improvements in chemotherapeutic regimens for children with acute lymphoblastic leukemia (ALL) have resulted in cure rates of~80%. In vivo and in vitro resistance to antileukemic agents are associated with a relatively poor prognosis [1] . However, still very little is known about the underlying genetic defects of drug resistance. Classic resistance mechanisms such as overexpression of multidrug resistance proteins MDR-1, MRP-1, MVP/ LRP and BCRP do not seem to play a major role in ALL [2] . Two genetic subclasses of ALL, i.e. those involving rearrangement of BCR-ABL and MLL are associated with a poor outcome but account only for a low number of therapy failures in absolute sense. The majority of therapy failures still occur in the large so-called favorable genetic subgroups such as hyperdiploid ALL and TEL-AML1 rearranged ALL. This illustrates that the current genetic classification of ALL is not sufficient and that the genes that contribute to therapy response or drug resistance are not known. When focusing on known candidate resistance genes, researchers are heavily limited by the knowledge they possess and new insights are difficult to obtain by studying these individual genes. Although gene expression profiling techniques have the disadvantage that they are "just fishing expeditions" the advantage is that the whole genome or at least a very large part of the genome is screened which may give us new ideas on genes that might be important for therapy failure and new drug targeting in ALL.

We have determined the in vitro sensitivity to 4 important drugs used in ALL treatment, i.e. prednisolone, vincristine, L-asparaginase and daunorubicin in 173 children with ALL and compared the gene expression profile of in vitro sensitive-and resistant cases [3] . Out of 14.500 probe sets we identified sets of genes differentially expressed in B-lineage ALL cases that were either sensitive or resistant to prednisolone (33 genes), vincristine (40 genes), L-asparaginase (35 genes) and daunorubicin (20 genes). Out of the total of 124 genes, 121 had never been linked to drug resistance before. A high combined gene expression score indicating resistance to the 4 drugs appeared to be significantly associated with a poor outcome: the hazard ratio for relapse was 3.0 for a high gene expression resistance score compared to a low score). This prognostic relevance was independent of all other known risk factors. Very important was the finding that the prognostic relevance of this gene expression score of resistance was confirmed in an independent population of patients treated in another institute with another protocol but including the 4 drugs mentioned. The hazard ratio for relapse for patients with a high score in this population was even 11.9.

Using partly the same set of data we also investigated the differential expression of 70 key apoptosis genes between drug sensitive and -resistant ALL cases [4] . No single apoptosis gene was related to resistance to all 4 unrelated drugs. Expression of MCL-1 and DAPK1 were associated with prednisolone resistance whereas BCL2L13, HRK and TNF were associated with resistance to L-asparaginase. Of these 5 genes only BCL2L13 overexpression predicted outcome significantly. This prognostic relevance appeared to be independent from all other known risk factors and was confirmed in an independent second group of patients treated with another protocol.

The above-mentioned studies do not identify the genes that have the strongest predictive value for outcome because the first selection was made on the basis of in vitro drug resistance phenotype. This leads to a selection bias and an underestimation of the prognostic power of gene expression profiling. It has been demonstrated that the different lineages of ALL (B-versus T-lineage) and the genetic subclasses of B-lineage ALL (TEL-AML1, BCR-ABL, hyperdiploid, E2A-PBX1, MLL) have specific gene expression profiles that can be recognize with an accuracy of more than 95%. This suggests that the underlying biology of these subtypes of ALL differs and in conjunction with this also their underlying resistance mechanisms and the gene expression profiles with predictive value for outcome. Indeed we showed that for instance the mechanism of L-Asparaginase differs between TEL-AML1 rearranged ALL and other genetic subtypes of ALL. In the latter the expression of asparagine synthetase was related to L-asparaginase resistance while in TEL-AML1 rearranged ALL this was not the case [5] . This also implies that it might be difficult to identify one single gene that predicts outcome in all subtypes of ALL. The group of Willman discovered a new gene that was named OPAL1 (outcome predictor in acute leukemia) of which a high expression was predictive of a favorable outcome in childhood ALL [6] . In two independent cohorts of patients including >400 cases we observed that a high expression of OPAL1 was associated with the favorable genetic subclass of TEL-AML1 as was also found by these authors. However we could not confirm the independent prognostic relevance of OPAL1 expression in the total groups of ALL patients nor in the genetic subgroups such as T-ALL and TEL/AML1 positive or -negative Blineage ALL [7] . The prognostic relevance of OPAL1 expression seems therefore therapy dependent or it might be difficult to reproduce gene expression findings from one laboratory to another. The fact however that the gene profiles of genetic subclasses of ALL are reproducible between different laboratories suggest that the first explanation may be more likely to be true.

An advantage of micro-array technologies is that they may lead to new and unexpected insights into the background of drug resistance and may lead to new genes or pathways that may serve as therapeutic targets. An important example is that our array analyses revealed that prednisolone resistance in B-lineage ALL is associated with an overexpression of genes involved in glycolysis, i.e. the glucose transporter 3 (GLUT3) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). In addition, prednisolone resistant cell lines were shown to have an increased glycolytic rate. The glycolysis inhibitor 2-deoxy-Dglucose (2-DG) sensitized prednisolone resistant but not-sensitive cell lines to prednisolone induced cell kill. This effect appeared to be specific for prednisolone and not for other drugs. Targeting the enhanced glycolysis in ALL may therefore be a suitable approach to modulate glucocorticoid resistance in ALL [8] .

A second example is the overexpression of MCL-1 in prednisolone resistant ALL cells. This was found not only in common/pre B-ALL but also in MLL gene rearranged infant ALL. Down-regulation of MCL-1 by RNAi sensitized ALL cells to glucocorticoid induced cell kill. Hence, inhibition of MCL-1 also offers an interesting therapeutic strategy in ALL.

Specific gene expression profiles are associated with resistance to different classes of antileukemic drugs. Profiles of genes associated with in vitro drug resistance have independent prognostic value. Analysis of pathways aberrantly expressed in resistant cells may lead to new therapeutic strategies in ALL.

Amphotericin B lipid soluble formulations vs amphotericin B in cancer patients with neutropenia

Amphotericin B versus fluconazole for controlling fungal infections in neutropenic cancer patients (Cochrane Review)

Antifungal prophylaxis for severely neutropenic chemotherapy recipients: a meta analysis of randomizedcontrolled clinical trials

Guide to development of practice guidelines

Invasive aspergillosis

Trends in mortality due to invasive mycotic diseases in the United States

Aspergillosis case-fatality rate: systematic review of the literature

Defining opportunistic invasive fungal infections in immunocompromised patients with cancer and hematopoietic stem cell transplants: an international consensus

Invasive aspergillosis in allogeneic stem cell transplant recipients: changes in epidemiology and risk factors

Invasive pulmonary aspergillosis in neutropenic patients during hospital construction: before and after chemoprophylaxis and institution of HEPA filters

Prophylaxis of invasive fungal infections in patients with hematological malignancies and solid tumors. Guidelines of the Infectious Diseases Working Party (AGIHO) of the German Society of Hematology and Oncology (DGHO)

Evidence-based assessment of primary antifungal prophylaxis in patients with hematologic malignancies

A controlled trial of fluconazole to prevent fungal infections in patients undergoing bone marrow transplantation

Efficacy and safety of fluconazole prophylaxis for fungal infections after marrow transplantation-a prospective, randomized, double-blind study

Prolonged fluconazole prophylaxis is associated with persistent protection against candidiasis-related death in allogeneic marrow transplant recipients: long-term follow-up of a randomized, placebo-controlled trial

Randomized placebo-controlled trial of fluconazole prophylaxis for neutropenic cancer patients: benefit based on purpose and intensity of cytotoxic therapy. The Canadian Fluconazole Prophylaxis Study Group

A randomized, double-blind, double-dummy, multicenter trial of voriconazole and fluconazole in the treatment of esophageal candidiasis in immunocompromised patients

Tolerability and safety of rituximab (MabThera)

Mechanism of action of therapeutic monoclonal antibodies

Renaissance of cancer therapeutic antibodies

Campath-1H) for treatment of lymphoid malignancies in the age of nonmyeloablative conditioning?

Campath-1H (anti-CD52) monoclonal antibody therapy in lymphoproliferative disorders

Targeting CD20 in Follicular NHL: Novel Anti-CD20 Therapies, Antibody Engineering, and the Use of Radioimmunoconjugates

An overview of the current clinical use of the anti-CD20 monoclonal antibody rituximab

CD33-directed therapy with gemtuzumab ozogamicin in acute myeloid leukemia: progress in understanding cytotoxicity and potential mechanisms of drug resistance

Mechanism of action and resistance to monoclonal antibody therapy

CD20: a gene in search of a function

Unique toxicities and resistance mechanisms associated with monoclonal antibody therapy

The CD52 antigen and development of the CAMPATH antibodies

CD52 (Campath1)

Manufacture and quality control of CAMPATH-1 antibodies for clinical trials

Phase I clinical trial using escalating single-dose infusion of chimeric anti-CD20 monoclonal antibody (IDEC-C2B8) in patients with recurrent B-cell lymphoma

IDEC-C2B8 (Rituximab) anti-CD20 monoclonal antibody therapy in patients with relapsed low-grade non-Hodgkin's lymphoma

Preclinical evaluation of 90Y-labeled anti-CD20 monoclonal antibody for treatment of non-Hodgkin's lymphoma

European public assessment report. Summary of product characteristics

European public assessment report. Summary of product characteristics

Rituximab (monoclonal anti-CD20 antibody): mechanisms of action and resistance

Functional role of lipid rafts in CD20 activity?

Cross-linking of the CAMPATH-1 antigen (CD52) mediates growth inhibition in human B-and T-lymphoma cell lines, and subsequent emergence of CD52-deficient cells

Anti-CD20-and B-cell receptor-mediated apoptosis: evidence for shared intracellular signaling pathways

Rituxan (anti-CD20 antibody)-induced translocation of CD20 into lipid rafts is crucial for calcium influx and apoptosis

Cellular and molecular signal transduction pathways modulated by rituximab (rituxan, anti-CD20 mAb) in non-Hodgkin's lymphoma: implications in chemosensitization and therapeutic intervention

Variation in gene expression patterns in follicular lymphoma and the response to rituximab

Prediction of survival in follicular lymphoma based on molecular features of tumor-infiltrating immune cells

Single agent rituximab in patients with follicular or mantle cell lymphoma: clinical and biological factors that are predictive of response and event-free survival as well as the effect of rituximab on the immune system: a study of the Swiss Group for

Biological and molecular characterization of PNH-like lymphocytes emerging after Campath-1H therapy

The PNH phenotype cells that emerge in most patients after CAMPATH-1H therapy are present prior to treatment

Influence of CD33 expression levels and ITIM-dependent internalization on gemtuzumab ozogamicin-induced cytotoxicity

Multidrug-resistance phenotype and clinical responses to gemtuzumab ozogamicin

The peripheral benzodiazepine receptor ligand PK11195 overcomes different resistance mechanisms to sensitize AML cells to gemtuzumab ozogamicin

High CD33-antigen loads in peripheral blood limit the efficacy of gemtuzumab ozogamicin (Mylotarg) treatment in acute myeloid leukemia patients

Adverse reactions: Rituximab, Alemtuzumab, Ibritumomab and Gemtuzumab

Unusual viral infections (progressive multifocal leukoencephalopathy and cytomegalovirus disease) after high-dose chemotherapy with autologous blood stem cell rescue and peritransplantation rituximab

Pure red cell aplasia due to parvovirus following treatment with CHOP and rituximab for B-cell lymphoma

Acute hepatitis B in a patient with antibodies to hepatitis B surface antigen who was receiving rituximab

N-ras mutations in adult de novo acute myelogenous leukemia: prevalence and clinical significance

Hematopoietic cell fate and the initiation of leukemic properties in primitive primary human cells are influenced by Ras activity and farnesyltransferase inhibition

Molecular aspects of chemical carcinogenesis: the roles of oncogenes and tumour suppressor genes

Small molecule FLT3 tyrosine kinase inhibitors

Targeting the Ras signaling pathway: a rational, mechanism-based treatment for hematologic malignancies?

The role of FLT3 in haematopoietic malignancies

Human somatic PTPN11 mutations induce hematopoietic-cell hypersensitivity to granulocyte-macrophage colony-stimulating factor

Sodium stibogluconate is a potent inhibitor of protein tyrosine phosphatases and augments cytokine responses in hemopoietic cell lines

JAK2 in myeloproliferative disorders is not just another kinase

Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell

A Quantitative Assay for the Number of Murine Lymphoma Cells Capable of Proliferation in Vivo

Similar MLL-associated leukemias arising from selfrenewing stem cells and short-lived myeloid progenitors

Primary bioassay of human tumor stem cells

Acute myeloid leukemia originates from a hierarchy of leukemic stem cell classes that differ in self-renewal capacity

MOZ-TIF2, but not BCR-ABL, confers properties of leukemic stem cells to committed murine hematopoietic progenitors

Unique molecular and cellular features of acute myelogenous leukemia stem cells

A cell initiating human acute myeloid leukaemia after transplantation into SCID mice

Stem cells in normal and leukemic hemopoiesis (Henry Stratton Lecture)

AML1/ETO-expressing nonleukemic stem cells in acute myelogenous leukemia with 8;21 chromosomal translocation

Mouse Myeloma Tumor Stem Cells: A Primary Cell Culture Assay

JunB deficiency leads to a myeloproliferative disorder arising from hematopoietic stem cells

Stem cells, cancer, and cancer stem cells

MLL-GAS7 transforms multipotent hematopoietic progenitors and induces mixed lineage leukemias in mice

Cancer stem cells: lessons from leukemia

Cancer mortality surveillance-United States

Acute myeloid leukemia and acute promyelocytic leukemia

On the value of intensive remission-induction chemotherapy in elderly patients of 65+ years with acute myeloid leukemia: a randomized phase III study of the European Organization for Research and Treatment of Cancer Leukemia Group

Use of glycosylated recombinant human G-CSF (lenograstim) during and/or after induction chemotherapy in patients 61 years of age and older with acute myeloid leukemia: final results of AML-13, a randomized phase 3 study of the European Organisation for Research and Treatment of Cancer and Gruppo Italiano Malattie Ematologiche dell'Adulto (EORTC/GIMEMA) Leukemia Groups

Efficacy and safety of gemtuzumab ozogamicin in patients with CD33-positive acute myeloid leukemia in first relapse

Final report of the efficacy and safety of gemtuzumab ozogamicin (Mylotarg) in patients with CD33-positive acute myeloid leukemia in first recurrence

Sequential administration of gemtuzumab ozogamicin and conventional chemotherapy as first line therapy in elderly patients with acute myeloid leukemia: a phase II study (AML-15) of the EORTC and GIMEMA leukemia groups

Outcome for children with relapsed acute myeloid leukaemia following initial therapy in the Medical Research Council (MRC) AML 10 trial. MRC Childhood Leukaemia Working Party

A simple, robust, validated and highly predictive index for the determination of risk-directed therapy in acute myeloid leukaemia derived from the MRC AML 10 trial. United Kingdom Medical Research Council's Adult and Childhood Leukaemia Working Parties

Immunophenotypic evidence of leukemia after induction therapy predicts relapse: results from a prospective Children's Cancer Group study of 252 patients with acute myeloid leukemia

Mortality in overweight and underweight children with acute myeloid leukemia

Glutathione S-transferase polymorphisms and outcome of chemotherapy in childhood acute myeloid leukemia

Glutathione S-transferase polymorphisms in children with myeloid leukemia: a Children's Cancer Group study

Down syndrome and leukemia, an update

Acute myeloid leukemia in children with Down syndrome

Acute myeloid leukemia (AML) in Down's syndrome is highly responsive to chemotherapy: experience on Pediatric Oncology Group AML Study 8498

Increased age at diagnosis has a significantly negative effect on outcome in children with Down syndrome and acute myeloid leukemia: a report from the Children's Cancer Group Study 2891

Treatment-related deaths during induction and first remission of acute myeloid leukaemia in children treated on the Tenth Medical Research Council acute myeloid leukaemia trial (MRC AML10). The MCR Childhood Leukaemia Working Party

Absence of reverse transcription-polymerase chain reaction detectable residual disease in patients with acute promyelocytic leukemia in long-term remission

The significance of minimal residual disease in patients with t (15;17)

Early detection of relapse by prospective reverse transcriptase-polymerase chain reaction analysis of the PML/RARalpha fusion gene in patients with acute promyelocytic leukemia enrolled in the GIMEMA-AIEOP multicenter

Persistence of the 8;21 translocation in patients with acute myeloid leukemia type M2 in long-term remission

Early immunophenotypical evaluation of minimal residual disease in acute myeloid leukemia identifies different patient risk groups and may contribute to postinduction treatment stratification

Immunological evaluation of minimal residual disease (MRD) in acute myeloid leukaemia (AML)

The importance of diagnostic cytogenetics on outcome in AML: analysis of 1,612 patients entered into the MRC AML 10 trial. The Medical Research Council Adult and Children's Leukaemia Working Parties

Relationships between age at diagnosis, clinical features, and outcome of therapy in children treated in the Medical Research Council AML 10 and 12 trials for acute myeloid leukemia

Marked improvements in outcome with chemotherapy alone in paediatric acute myeloid leukemia: results of the United Kingdom Medical Research Council's 10th AML trial. MRC Childhood Leukaemia Working Party

Chromosomal abnormalities in 478 children with acute myeloid leukemia: clinical characteristics and treatment outcome in a cooperative pediatric oncology group study-POG 8821

The predictive value of hierarchical cytogenetic classification in older adults with acute myeloid leukemia (AML): analysis of 1065 patients entered into the United Kingdom Medical Research Council AML11 trial

Clinical and prognostic significance of chromosomal abnormalities in childhood acute myeloid leukemia de novo

Acute myeloid leukemia with t (6;9) (p23;q34): association with myelodysplasia, basophilia, and initial CD34 negative immunophenotype

The translocation (6;9), associated with a specific subtype of acute myeloid leukemia, results in the fusion of two genes, dek and can, and the expression of a chimeric, leukemia-specific dek-can mRNA

The translocation (6;9) (p23;q34) shows consistent rearrangement of two genes and defines a myeloproliferative disorder with specific clinical features

Dek-can rearrangement in translocation (6;9)(p23;q34)

Favorable impact of the t(9;11) in childhood acute myeloid leukemia

Prognostic implication of FLT3 and N-RAS gene mutations in acute myeloid leukemia

Activating mutation of D835 within the activation loop of FLT3 in human hematologic malignancies

FLT3 in human hematologic malignancies

Internal tandem duplication of the FLT3 gene is a novel modality of elongation mutation which causes constitutive activation of the product

Prevalence and prognostic significance of Flt3 internal tandem duplication in pediatric acute myeloid leukemia

Prognostic factors and risk-based therapy in pediatric acute myeloid leukemia

Activating mutations of RTK/ras signal transduction pathway in pediatric acute myeloid leukemia

FLT3, RAS, and TP53 mutations in elderly patients with acute myeloid leukemia

Impact of FLT3 mutations and promyelocytic leukaemia-breakpoint on clinical characteristics and prognosis in acute promyelocytic leukaemia

Relationship between FLT3 mutation status, biologic characteristics, and response to targeted therapy in acute promyelocytic leukemia

FLT3-activating mutations in acute promyelocytic leukaemia: a rationale for risk-adapted therapy with FLT3 inhibitors

Analysis of FLT3-activating mutations in 979 patients with acute myelogenous leukemia: association with FAB subtypes and identification of subgroups with poor prognosis

FLT3-ITD and tyrosine kinase domain mutants induce 2 distinct phenotypes in a murine bone marrow transplantation model

Definition of a standard-risk group in children with AML

Intensively timed induction therapy followed by autologous or allogeneic bone marrow transplantation for children with acute myeloid leukemia or myelodysplastic syndrome: a Childrens Cancer Group pilot study

Activating and dominant inactivating c-KIT catalytic domain mutations in distinct clinical forms of human mastocytosis

FMS mutations in myelodysplastic, leukemic, and normal subjects

FMS and p53 mutations and poor clinical outcome in myelodysplasias: a 10-year follow-up

Mutations in KIT and RAS are frequent events in pediatric core-binding factor acute myeloid leukemia

KIT activating mutations: incidence in adult and pediatric acute myeloid leukemia, and identification of an internal tandem duplication

Activating mutations of c-kit at codon 816 confer drug resistance in human leukemia cells

Signal transducer and activator of transcription 3 activation is required for Asp(816) mutant c-Kit-mediated cytokineindependent survival and proliferation in human leukemia cells

The presence of a FLT3 internal tandem duplication in patients with acute myeloid leukemia (AML) adds important prognostic information to cytogenetic risk group and response to the first cycle of chemotherapy: analysis of 854 patients from the United Kingdom Medical Research Council AML 10 and 12 trials

FLT3 internal tandem duplication in 234 children with acute myeloid leukemia: prognostic significance and relation to cellular drug resistance

Favorable prognostic significance of CEBPA mutations in patients with de novo acute myeloid leukemia: a study from the Acute Leukemia French Association (ALFA)

Dominant-negative mutations of CEBPA, encoding CCAAT/enhancer binding protein-alpha (C/EBPalpha), in acute myeloid leukemia

Elevated expression of the AF1q gene, an MLL fusion partner, is an independent adverse prognostic factor in pediatric acute myeloid leukemia

Nucleophosmin gene mutations are predictors of favorable prognosis in acute myelogenous leukemia with a normal karyotype

Mutant nucleophosmin (NPM1) predicts favorable prognosis in younger adults with acute myeloid leukemia and normal cytogenetics: interaction with other gene mutations

Prevalence, clinical profile, and prognosis of NPM mutations in AML with normal karyotype

Mutations in nucleophosmin (NPM1) in acute myeloid leukemia (AML): association with other gene abnormalities and previously established gene expression signatures and their favorable prognostic significance

Telomerase activity is prognostic in pediatric patients with acute myeloid leukemia: comparison with adult acute myeloid leukemia

Vascular endothelial growth factor secretion is an independent prognostic factor for relapsefree survival in pediatric acute myeloid leukemia patients

High levels of Wilms' tumor gene (wt1) mRNA in acute myeloid leukemias are associated with a worse long-term outcome

Real-time quantitative PCR detection of WT1 gene expression in children with AML: prognostic significance, correlation with disease status and residual disease detection by flow cytometry

Prognostic implications of Wilms' tumor gene (WT1) expression in patients with de novo acute myeloid leukemia

Prognostic impact of RT-PCR-based quantification of WT1 gene expression during MRD monitoring of acute myeloid leukemia

Gene expression profiling in acute myeloid leukemia

Gene expression profiles at diagnosis in de novo childhood AML patients identify FLT3 mutations with good clinical outcomes

Identification of a gene expression signature associated with pediatric AML prognosis

Frequency of prolonged remission duration after high-dose cytarabine intensification in acute myeloid leukemia varies by cytogenetic subtype

Recombinant human granulocyte-macrophage colony-stimulating factor after chemotherapy in patients with acute myeloid leukemia at higher age or after relapse

The value of allogeneic bone marrow transplant in patients with acute myeloid leukaemia at differing risk of relapse: results of the UK MRC AML 10 trial

Therapierealisierung und Komplikationen in der Therapiestudie BFM-83 für die akute myeloische Leukämie

Idarubicin improves blast cell clearance during induction therapy in children with AML: results of study AML-BFM 93

Improved treatment results in high-risk pediatric acute myeloid leukemia patients after intensification with high-dose cytarabine and mitoxantrone: results of Study Acute Myeloid Leukemia-Berlin-Frankfurt-Münster 93

Early deaths and treatment-related mortality in children undergoing therapy for acute myeloid leukemia: analysis of the multicenter clinical trials AML-BFM 93 and AML-BFM 98

Antileukemic effect of chronic graft-versus-host disease: contribution to improved survival after allogeneic marrow transplantation

Graft-versus-leukemia effect of donor lymphocyte transfusions in marrow grafted patients. European Group for Blood and Marrow Transplantation Working Party Chronic Leukemia

Acute myeloid leukemia

Acute myeloid leukemia: treatment over 60

Early allogeneic blood stem cell transplantation after modified conditioning therapy during marrow aplasia: stable remission in high-risk acute myeloid leukemia

Experience with gemtuzumab ozogamicin ("mylotarg") + all-trans retinoic acid in untreated acute promyelocytic leukemia

Gentuzumab ozogamicin (''mylotarg'') as a single agent for molecularly relapsed acute promyelocytic leukemia

Potential curability of newly diagnosed acute promyelocytic leukemia without chemotherapy: the example of liposmal all-trans retinoic acid

Definition of relapse risk and role of nonanthracycline drugs for consolidation in patients with acute promyelocytic leukemia: a joint study of the PETHEMA and GIMEMA cooperative groups

Use of arsenic trioxide in acute promyelocytic leukemia leads to cardiac toxicity in African-American patients

Imatinib mesylate therapy may overcome the poor prognostic significance of deletions of derivative chromosome 9 in patients with chronic myelogenous leukemia

Presenting white blood cell count and kinetics of molecular remission predict prognosis in acute promyelocytic leukemia treated with all-trans retinoic acid: results of the randomized MRC trial

All-transretinoic acid/As2O3 combination yields a high quality remission and survival in newly diagnosed acute promyelocytic leukemia

All-trans retinoic acid and anthracycline monochemotherapy for the treatment of elderly patients with acute promyelocytic leukemia

Treatment of older adults with acute promyelocytic leukaemia

The importance of molecular monitoring in acute promyelocytic leukaemia

Riskadapted treatment of acute promyelocytic leukemia with all-trans-retinoic acid and anthracycline monochemotherapy: a multicenter study by the PETHEMA group

Identificaton of a translocation with quinacrine fluorescence in a patient with acute leukemia

Molecular genetics in acute leukemia

Acute myeloid leukemia with t(8;21)/AML1/ ETO: a distinct biological and clinical entity

Prognostic Impact of C-Kit Mutations in Core Binding Factor-Leukemia

Incidence and Prognosis of RTKs and RAS Mutations in CBF AML. A Retrospective Study of French Adult ALFA and Pediatric LAME Trials

Expression of a conditional AML1-ETO oncogene bypasses embryonic lethality and establishes a murine model of human t(8;21) acute myeloid leukemia

AML1-ETO expression is directly involved in the development of acute myeloid leukemia in the presence of additional mutations

Hematopoietic stem cell expansion and distinct myeloid developmental abnormalities in a murine model of the AML1-ETO translocation

Stem cell expression of the AML1/ETO fusion protein induces a myeloproliferative disorder in mice

An activated receptor tyrosine kinase, TEL/PDGFbetaR, cooperates with AML1/ETO to induce acute myeloid leukemia in mice

Oncogenic transcription factors in the human acute leukemias

Molecular genetics of human leukemias: new insights into therapy

The AML1-ETO fusion gene and the FLT3 length mutation collaborate in inducing acute leukemia in mice

Mutations in KIT and RAS are frequent events in pediatric core-binding factor acute myeloid leukemia

AML1-ETO and C-KIT mutation/ overexpression in t(8;21) leukemia: implication in stepwise leukemogenesis and response to Gleevec

KIT-D816 mutations in AML1-ETO positive AML are associated with impaired event-free and overall survival

Drug therapy for acute myeloid leukemia

Angiogenesis in cancer, vascular, rheumatoid and other disease

Mechanisms of angiogenesis

Role of angiogenesis inhibitors in acute myeloid leukemia

Isolation of angiopoietin-1, a ligand for the TIE2 receptor, by secretion-trap expression cloning

The angiopoietins. Yin and Yang in angiogenesis

Angiopoietins 3 and 4: diverging gene counterparts in mice and humans

Distinct roles of the receptor tyrosine kinases Tie-1 and Tie-2 in blood vessel formation

Signaling angiogenesis and lymphangiogenesis

Angiopoietin-2, a natural antagonist for Tie2 that disrupts in vivo angiogenesis

From the Cover: Angiopoietin-2 displays VEGF-dependent modulation of capillary structure and endothelial cell survival in vivo

Vessel cooption, regression, and growth in tumors mediated by angiopoietins and VEGF

Orchestration of angiogenesis and arteriovenous contribution by angiopoietins and vascular endothelial growth factor (VEGF)

Analysis Of Concerted Expression Of Angiogenic Growth Factors In Acute Myeloid Leukemia: Expression Of Angiopoietin-2

Represents An Independent Prognostic Factor For Overall Survival

In vivo administration of vascular endothelial growth factor (VEGF) and its antagonist, soluble neuropilin-1, predicts a role of VEGF in the progression of acute myeloid leukemia in vivo

Antiangiogenic treatment with endostatin inhibits progression of AML in vivo

A phase 2 clinical study of SU5416 in patients with refractory acute myeloid leukemia

Pregnancy induces minor Histocompatibility antigen specific cytotoxic T cells: implications for stem cell transplantation and immunotherapy

HA-1-specific regulator and effector CD8+ T cells, and HA-1 microchimerism, in allograft tolerance

Cord blood comprises antigen-experienced T cells specific for maternal minor histocompatibility antigen HA-1. B. Mommaas

Immunotherapy of cancer through targeting of minor histocompatibility antigens

Human cytotoxic T lymphocytes specific for a single minor histocompatibility antigen HA-1 are effective against human lymphoblastic leukaemia in NOD/scid mice

Vitamin A and/or high-dose Ara-C in the maintenance of remission in acute myelogenous leukaemia in children?

A population-based study of 272 children with acute myeloid leukaemia treated on two consecutive protocols with different intensity: best outcome in girls, infants, and children with Down's syndrome. Nordic Society of Paediatric Haematology and Oncology (NOPHO)

Treatment stratification based on initial in vivo response in acute myeloid leukaemia in children without Down syndrome. Results of NOPHO-AML trials

Cytogenetic abnormalities in childhood acute myeloid leukaemia: A Nordic series comprising all children enrolled in the NOPHO-93-AML trial between 1993 and

Long-term results in children with AML: NOPHO-AML Study Groupreport of three consecutive trials

Acute leukaemia in children with Down syndrome: a population-based Nordic study

Down syndrome and acute myelogenous leukemia. A population based study in the five Nordic countries

A pediatric approach to the WHO classification of myelodysplastic and myeloproliferative diseases

Optimal treatment intensity in children with Down syndrome and myeloid leukaemia: data from 56 children treated on NOPHO-AML protocols and a review of the literature

Targeting the multidrug resistance-1 transporter in AML: molecular regulation and therapeutic strategies

Mitoxantrone, etoposide, and cytarabine with or without valspodar in patients with relapsed or refractory acute myeloid leukemia and high-risk myelodysplastic syndrome: a phase III trial (E2995)

Phase 3 study of the multidrug resistance modulator PSC-833 in previously untreated patients 60 years of age and older with acute myeloid leukemia: Cancer and Leukemia Group B Study 9720

The value of the MDR1 reversal agent PSC-833 in addition to daunorubicin and cytarabine in the treatment of elderly patients with previously untreated acute myeloid leukemia (AML), in relation to MDR1 status at diagnosis

Treatment Failure Is Strongly Predicted by P-Glycoprotein (Pgp) Function but Not by Multidrug Resistance Protein (MRP-1), Breast Cancer Resistance Protein (BCRP) or Lung Resistance Protein (LRP) in Acute Myeloid Leukemia (AML) Patients 60 Years and Older Receiving Intensive Chemotherapy (CALGB 9720/9760)

Dose Escalation Studies of Cytarabine, Daunorubicin, and Etoposide With and Without Multidrug Resistance Modulation With PSC-833 in Untreated Adults With Acute Myeloid Leukemia Younger Than 60 Years: Final Induction Results of Cancer and Leukemia Group B Study 9621

A Randomized Comparison of Induction Therapy for Untreated Acute Myeloid Leukemia (AML) in Patients < 60 Years Using P-Glycoprotein (Pgp) Modulation with Valspodar (PSC833): Preliminary Results of Cancer and Leukemia Group B Study 19808. Session Type: Oral Session

Parallel phase I studies of daunorubicin given with cytarabine and etoposide with or without the multidrug resistance modulator PSC-833 in previously untreated patients 60 years of age or older with acute myeloid leukemia: results of cancer and leukemia group B study 9420

Benefit of cyclosporine modulation of drug resistance in patients with poor-risk acute myeloid leukemia: a Southwest Oncology Group study

Clinical effects and P-glycoprotein inhibition in patients with acute myeloid leukemia treated with zosuquidar trihydrochloride, daunorubicin and cytarabine

Simultaneous activity of MRP1 and Pgp is correlated with in vitro resistance to daunorubicin and with in vivo resistance in adult acute myeloid leukemia

Multidrug resistant cells with high proliferative capacity determine response to therapy in acute myeloid leukemia

Acute lymphoblastic leukemia in the elderly: the Edouard Herriot Hospital experience

Subtypes and treatment outcome in adult acute lymphoblastic leukemia less than or greater than 55 years

Acute lymphoblastic leukemia in elderly: the Polish Adult Leukemia Group experience

Cytogenetics adds independent prognostic information in adults with acute lymphoblastic leukaemia on MRC trial UKALL XA

Cytogenetic abnormalities in adult acute lymphoblastic leukemia: correlations with hematologic findings and outcome

Prospective karyotype analysis in adult acute lymphoblastic Leukemia. The Cancer and Leukemia Group B experience

Impact of age on the biology of acute leukemia

Results of treatment with hyper-CVAD, a dose-intensive regimen, for adult acute lymphoblastic leukemia

Enrollment of elderly patients in clinical trials for cancer drug registration: a 7-year experience by the US Food and Drug Administration

The impact of age on outcome in lymphoblastic leukaemia

a report from the MRC Paediatric and Adult Working Parties

A five-drug remission induction regimen with intensive consolidation for adults with acute lymphoblastic leukemia: Cancer and Leukemia Group B study 8811

A randomized controlled trial of filgrastim during remission induction and consolidation chemotherapy for adults with acute lymphoblastic leukemia: CALGB study 9111

Results of a shortened, dose reduced treatment protocol in elderly patients with acute lymphoblastic leukemia

Subtype adjusted therapy improves outcome of elderly patients with acute lymphoblastic leukemia

Treatment of elderly patients with acute lymphoblastic leukemia

The treatment of acute lymphoblastic leukaemia in the elderly

Acute lymphoblastic leukemia in the elderly. A twelve-year retrospective

Treatment of Philadelphia chromosome positive acute lymphoblastic leukemia

Leading prognostic relevance of the BCR-ABL translocation in adult acute B-lineage lymphoblastic leukemia: a prospective study of the German Multicenter Trial Group

A phase 2 study of imatinib in patients with relapsed or refractory Philadelphia chromosome-positive acute lymphoid leukemias

Update of the hyper-CVAD and imatinib mesylate regimen in Philadelphia (Ph) positive acute lymphocytic leukemia

Combination of intensive chemotherapy and imatinib can rapidly induce high-quality complete remission for a majority of patients with newly diagnosed BCR-ABL-positive acute lymphoblastic

Imatinib given concurrently with induction chemotherapy is superior to imatinib subsequent to induction and consolidation in newly diagnosed Philadelphiapositive acute lymphoblastic leukemia

Dramatic improvement in CR rate and CR duration with imatinib in adult and elderly Ph+ ALL patients: results of the GIMEMA prospective study LAL0201

A randomized phase II study comparing imatinib with chemotherapy as induction therapy in elderly patients with newly diagnosed Philadelphia-positive acute lymphoid leukemias

High-dose daunorubicin as liposomal compound (Daunoxome) in elderly patients with acute lymphoblastic leukemia

Double induction strategy for acute myeloid leukemia. The effect of high-dose cytarabine with mitoxantrone instead of standard-dose cytarabine with daunorubicin and 6-thioguanine. A randomized trial by the German AML Cooperative Group

For the German acute myeloid leukemia cooperative Group (1997) Effects of high dose cytarabin (HD-ara-C) as part of double induction strategy in acute promyelocytic leukemia (APL) and results of combination with all-trans retinoic acid (ATRA)

Bü chner T (2000) Double induction strategy including high dose cytarabine in combination with all-trans retinoic acid: Effects in patients with newly diagnosed acute promyelocytic leukemia

Uncertain role of increased intensity chemotherapy with high-dose cytarabine in acute promyelocytic leukemia

Treatment of newly diagnosed acute promyelocytic leukemia: The impact of high dose ara-C

Treatment of Relapsed Acute Promyelocytic Leukemia

Risk-adapted treatment of acute promyelocytic leukemia with all-trans-retinoic acid and anthracycline monochemotherapy: a multicenter study by the PETHEMA group

Arsenic trioxide in the treatment of acute promyelocytic leukemia. A review of current evidence

Imatinib in Combination with Chemotherapy for Newly Diagnosed BCR/ ABL-positive Acute Lymphoblastic Leukemia in Adults R. OHNO, M. YANADA, and T. NAOE for Japan Adult Leukemia Study Group

Infections caused by viridans streptococci in patients with neutropenia

Levofloxacin to prevent bacterial infection in patients with cancer and neutropenia

Antibacterial prophylaxis after chemotherapy for solid tumors and lymphomas

A Predominantly Clonal Multi-Institutional Outbreak of Clostridium difficile-Associated Diarrhea with High Morbidity and Mortality

Role of glycopeptides as part of initial empirical treatment of febrile neutropenic patients: a meta-analysis of randomised controlled trials

Additional anti-Gram-positive antibiotic treatment for febrile neutropenic cancer patients

Time to clinical response: an outcome of antibiotic therapy of febrile neutropenia with implications for quality and cost of care

guidelines for the use of antimicrobial agents in neutropenic patients with cancer

Treatment of fungal infections in hematology and oncologyguidelines of the Infectious Diseases Working Party (AGIHO) of the German Society of Hematology and Oncology (DGHO)

Increasing volume and changing characteristics of invasive pulmonary aspergillosis on sequential thoracic computed tomography scans in patients with neutropenia

Total Therapy Study XV for Newly Diagnosed Childhood Acute Lymphoblastic Leukemia: Study Design and Preliminary Results C

Acute lymphoblastic leukemia

Childhood acute lymphoblastic leukaemia-current status and future perspectives

Extended follow-up of longterm survivors of childhood acute lymphoblastic leukemia

Moving towards individualized medicine with pharmacogenomics

Rationale and design of Total Therapy study XV for newly diagnosed childhood acute lymphoblastic leukemia

Acute lymphoblastic leukemia

New definition of remission in childhood acute lymphoblastic leukemia

Minimal residual disease in leukaemia patients

Determination of minimal residual disease in leukaemia patients

Pharmacogenetics of outcome in children with acute lymphoblastic leukemia

Karyotypic abnormalities create discordance of germline genotype and cancer cell phenotypes

Blast cell methotrexate-polyglutamate accumulation in vivo differs by lineage, ploidy, and methotrexate dose in acute lymphoblastic leukemia

International childhood acute lymphoblastic leukemia workshop

Folate pathway gene expression differs in subtypes of acute lymphoblastic leukemia and influences methotrexate pharmacodynamics

Results of therapy for acute lymphoblastic leukemia in black and white children

Concepts in use of high-dose methotrexate therapy

Intrinsic and acquired resistance to methotrexate in acute leukemia

Treatment-specific changes in gene expression discriminate in vivo drug response in human leukemia cells

Identification of genes associated with chemotherapy crossresistance and treatment response in childhood acute lymphoblastic leukemia

Conventional compared with individualized chemotherapy for childhood acute lymphoblastic leukemia

Minimal residual disease status before allogeneic bone marrow transplantation is an important determinant of successful outcome for children and adolescents with acute lymphoblastic leukemia

Minimal residual disease (MRD) status prior to allogeneic stem cell transplantation is a powerful predictor for post-transplant outcome in children with ALL

Minimal residual disease prior to stem cell transplant for childhood acute lymphoblastic leukaemia

Improved outcome in high-risk childhood acute lymphoblastic leukemia defined by prednisone-poor response treated with double Berlin-Frankfurt-Muenster protocol II

Double-delayed intensification improves event-free survival for children with intermediate-risk acute lymphoblastic leukemia: a report from the Children's Cancer Group

Prognostic importance of 6-mercaptopurine dose intensity in acute lymphoblastic leukemia

Mercaptopurine therapy intolerance and heterozygosity at the thiopurine S-methyltransferase gene locus

Extended follow-up of long-term survivors of childhood acute lymphoblastic leukemia

Low leukocyte counts with blast cells in cerebrospinal fluid of children with newly diagnosed acute lymphoblastic leukemia

Prognostic significance of cerebrospinal fluid (CSF) lymphoblasts (LB) at diagnosis (dx) in children with acute lymphoblastic leukemia (ALL)

Long-term results of three randomized trials (58831, 58832, 58881) in childhood acute lymphoblastic leukemia: a CLCG-EORTC report

Effect of initial central nervous system (CNS) status on event-free survival (EFS) in children and adolescents with acute lymphoblastic leukemia (ALL)

Diagnostic cerebrospinal fluid examination in children with acute lymphoblastic leukemia: significance of low leukocyte counts with blasts or traumatic lumbar puncture

Traumatic lumbar puncture at diagnosis adversely affects outcome in childhood acute lymphoblastic leukemia

Prognostic significance of blasts in the cerebrospinal fluid without pleiocytosis or a traumatic lumbar puncture in children with acute lymphoblastic leukemia: the experience of the Dutch Childhood Oncology Group

Improved outcome for children with acute lymphoblastic leukemia: results of Total Therapy Study XIIIB at St Jude Children's Research Hospital

Quantitative assessment of cerebral atrophy during and after treatment in children with acute lymphoblastic leukemia

Chemotherapy for acute lymphoblastic leukemia may cause subtle changes of the spinal cord detectable by somatosensory evoked potentials

Visual and verbal shortterm memory deficits in childhood leukemia survivors after intrathecal chemotherapy

Risk factors for traumatic and bloody lumbar puncture in children with acute lymphoblastic leukemia

Rationale and design of total therapy study XV for newly diagnosed childhood acute lymphoblastic leukemia

Treatment of acute lymphoblastic leukemia

TGF-(beta)1 maintains hematopoietic immaturity by a reversible negative control of cell cycle and induces CD34 antigen upmodulation

Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell

Abnormal TGFbeta levels in the amniotic fluid of Down syndrome pregnancies

Gatekeeper pathways and cellular background in the pathogenesis and therapy of AML

Transcriptional regulation of erythropoiesis: an affair involving multiple partners

Distinct patterns of hematopoietic stem cell involvement in acute lymphoblastic leukemia

Genomic profiling identifies alterations in TGFbeta signaling through loss of TGFbeta receptor expression in human renal cell carcinogenesis and progression

RUNX1 and GATA-1 coexpression and cooperation in megakaryocytic differentiation

Differences in the prevalence of PTPN11 mutations in FAB M5 paediatric acute myeloid leukaemia

Mutations in KIT and RAS are frequent events in pediatric core-binding factor acute myeloid leukemia

Origins of chromosome translocations in childhood leukaemia

Recent insights into the mechanism of myeloid leukemogenesis in Down syndrome

GATA1-a player in normal and leukemic megakaryopoiesis

Immunophenotypic differences between diagnosis and relapse in childhood AML: Implications for MRD monitoring

Developmental stage-selective effect of somatically mutated leukemogenic transcription factor GATA1

Enforced expression of the GATA-2 transcription factor blocks normal hematopoiesis

Transforming growth factor-beta1 causes transcriptional activation of CD34 and preserves haematopoietic stem/progenitor cell activity

Chromosome 21 encoded RUNX1 and ETS-2 overexpression in regenerating hematopoiesis in children with Down syndrome -implications in leukemiogenesis

Identification of BMP and activin membrane-bound inhibitor (BAMBI), an inhibitor of transforming growth factor-beta signaling, as a target of the betacatenin pathway in colorectal tumor cells

Aberrant protein expression of transcription factors BACH1 and ERG, both encoded on chromosome 21, in brains of patients with Down syndrome and Alzheimer's disease

The immunophenotypic profile of hepatic hemopoiesis in fetuses with Down's syndrome during the second trimester of development

Monitoring of minimal residual disease (MRD) by real-time quantitative reverse transcription PCR (RQ-RT-PCR) in childhood acute myeloid leukemia with AML1/ETO rearrangement

Acquired mutations in GATA1 in the megakaryoblastic leukemia of Down syndrome

FLT3 internal tandem duplication in 234 children with acute myeloid leukemia: prognostic significance and relation to cellular drug resistance

Clinical Trials for Childhood Acute Myeloid Leukemia at St. Jude Children's Research Hospital

RUBNITZ1,5 1Department of Hematology-Oncology, 2International Outreach Program, 3Department of Biostatistics, and 4Department of Pharmaceutical Sciences, St. Jude Children's Research Hospital; 5Department of Pediatrics

Gene-marking to trace origin of relapse after autologous bone-marrow transplantation

Clinical significance of residual disease during treatment in childhood acute myeloid leukaemia

Interim comparison of a continuous infusion versus a short daily infusion of cytarabine given in combination with cladribine for pediatric acute myeloid leukemia

Allogeneic bone marrow transplantation in a program of intensive sequential chemotherapy for children and young adults with acute nonlymphocytic leukemia in first remission

Early intensification of chemotherapy for childhood acute nonlymphoblastic leukemia: improved remission induction with a five-drug regimen including etoposide

Prognostic importance of cytogenetic subgroups in de novo pediatric acute nonlymphocytic leukemia

Pediatric acute myeloid leukemia: international progress and future directions Leukemia

Experience with 2-chlorodeoxyadenosine in previously untreated children with newly diagnosed acute myeloid leukemia and myelodysplastic diseases

Successive clinical trials for childhood acute myeloid leukemia at St Jude Children's Research Hospital

Favorable impact of the t(9;11) in childhood acute myeloid leukemia

Improved Remission Induction Rate of Childhood AML: Preliminary Results of the AML02 Trial

Acute promyelocytic leukaemia: evolving therapeutic strategies

Choice of chemotherapy in induction, consolidation and maintenance in acutepromyelocytic leukemia

Coco on behalf of the European APL Group of Experts. Arsenic trioxide in the treatment of acute promyelocytic leukemia. A review of current evidence

Front-line treatment of acute promyelocytic leukemia with AIDA induction followed by risk-adapted consolidation: results of the AIDA-2000 trial of the Italian GIMEMA group

Risk-adapted treatment of acute promyelocytic leukemia with all-trans retinoic acid and anthracycline monochemotherapy: a multicenter study by the PETHEMA Group

Definition of relapse risk and role of nonanthracycline drugs for consolidation in patients with acute promyelocytic leukemia: a joint study of the PETHEMA and GIMEMA cooperative groups

Risk-Adapted Treatment of Acute Promyelocytic Leukemia: Updated Results of the Spanish PETHEMA LPA99

Is ARAC Required in the Treatment of Newly Diagnosed APL? Results of a Randomized Trial

Granulocyte-macrophage colony-stimulating factor after initial chemotherapy for elderly patients with primary acute myelogenous leukemia

A randomized placebo-controlled phase III study of granulocyte-macrophage colonystimulating factor in adult patients (> 55 to 70 years of age) with acute myelogenous leukemia: a study of the Eastern Cooperative Oncology Group (E1490)

Use of recombinant GM-CSF during and after remission induction chemotherapy in patients aged 61 years and older with acute myeloid leukemia: final report of AML-11, a phase III randomized study of the Leukemia Cooperative Group of European Organisation for the Research and Treatment of Cancer and the Dutch Belgian Hemato

A double-blind placebo-controlled trial of granulocyte colony-stimulating factor in elderly patients with previously untreated acute myeloid leukemia: a Southwest oncology group study (9031)

Phase III study of the multidrug resistance modulator PSC-833 in previously untreated patients 60 years of age and older with acute myeloid leukemia: Cancer and Leukemia Group B study 9720

Postremission therapy in older patients with de novo acute myeloid leukemia: a randomized trial comparing mitoxantrone and intermediate-dose cytarabine with standard-dose cytarabine

Use of glycosylated recombinant human G-CSF (lenograstim) during and/or after induction chemotherapy in patients 61 years of age and older with acute myeloid leukemia: final results of AML-13, a randomized phase-3 study

Modification or Dose or Treatment Duration Has No Impact on Outcome of AML in Older Patients: Preliminary Results of the UK NCRI AML14 Trial

The importance of diagnostic cytogenetics on outcome in AML: analysis of 1,612 patients entered into the MRC AML 10 trial. The Medical Research Council Adult and Children's Leukaemia Working Parties

Karyotypic analysis predicts outcome of preremission and postremission therapy in adult acute myeloid leukemia: a Southwest Oncology Group/Eastern Cooperative Oncology Group Study

Karyotype is an independant prognostic parameter in therapy-related acute myeloid leukemia (t-AML): an analysis of 93 patients with t-AML in comparision to 1092 patients with de novo AML

Effect of time to complete remission on subsequent survival and disease-free survival time in AML, RAEB-t, and RAEB

Early blast clearance by remission induction chemotherapy is a major independant prognostic factor for both achievement of complete remission and long-term outcome in acute myeloid leukemia: data from the German AML cooperative group (AMLCG) 1992 trial

Treatment of refractory AML

Stage-specific application of allogeneic and autologous marrow transplantation in the management of acute myeloid leukemia

Who should be transplanted for AML

The search for optimal treatment in relapsed and refractory acute myeloid leukemia

Bone marrow transplants may cure patients with acute leukemia never achieving remission with chemotherapy

Outcome after allogeneic bone marrowe transplantfor leukemia in older adults

Unrelated bone marrow transplantation in patients with myelodysplastic syndromes and secondary acute myeloid leukemia: an EBMT survey. European Blood and Marrow Transplantation Group

Allogeneic transplantation from HLA-matched sibling or partially HLA-mismatched related donors for primary refractory acute leukemia

Long-term outcome after allogeneic hematopoietic stem cell transplantation for advanced stage acute myeloblastic leukemia: a retrospective study of 379 patients reported to the Societe Francaise de Greffe de Moelle (SFGM)

Treatment of murine leukemia with X-rays and homologous bone marrow

Chemotherapy compared with autologous or allogeneic bone marrow transplantation in the management of acute myeloid leukemia in first remission

Nonmyeloablative stem cell transplantation and cell therapy as an alternative to conventional bone marrow transplantation with lethal cytoreduction for the treatment of malignant and nonmalignant hematologic diseases

Transplant-lite: induction of graftversus-malignancy using fludarabine-based nonablative chemotherapy and allogeneic blood progenitor-cell transplantation as treatment for lymphoid malignancies

Melphalan and purine analog-containing preparative regimens: reduced-intensity conditioning for patients with hematologic malignancies undergoing allogeneic progenitor cell transplantation

Hematopoietic cell transplantation in older patients with hematologic malignancies: replacing highdose cytotoxic therapy with graft-versus-tumor effects

Low-dose total body irradiation (TBI) and fludarabine followed by hematopoietic cell transplantation (HCT) from HLA-matched or mismatched unrelated donors and postgrafting immunosuppression with cyclosporine and mycophenolate mofetil (MMF) can induce durable complete chimerism and sustained remissions in patients with hematological diseases

Nonablative versus reduced-intensity conditioning regimens in the treatment of acute myeloid leukemia and high-risk myelodysplastic syndrome: dose is relevant for long-term disease control after allogeneic hematopoietic stem cell transplantation

Non-myeloablative stem cell transplantation in AML, ALL and MDS: Disappointing outcome for patients with advanced disease

Non-myeloablative allografting from human leucocyte antigen-identical sibling donors for treatment of acute myeloid leukaemia in first complete remission

Fludarabine, cytarabine, G-CSF and idarubicin (FLAG-IDA) for the treatment of poor-risk myelodysplastic syndromes and acute myeloid leukaemia

An effective ageunrestricted m-AMSA-based second-line regimen for poor prognosis acute myeloid leukaemia

Sequential regimen of chemotherapy, reduced-intensity conditioning for allogeneic stem-cell transplantation, and prophylactic donor lymphocyte transfusion in high-risk acute myeloid leukemia and myelodysplastic syndrome

Allogeneic stem-cell transplantation from related and unrelated donors in older patients with myeloid leukemia

Durable remissions of myelodysplastic syndrome and acute myeloid leukemia after reduced-intensity allografting

Reduced-intensity conditioning for unrelated donor hematopoietic stem cell transplantation as treatment for myeloid malignancies in patients older than 55 years

Long lasting remissions in high risk AML and MDS following sequential therapy with chemotherapy, reduced intensity conditioning for allogeneic transplantation, and prophylactic DLT

Evidence for a graft-versusleukemia effect after allogeneic peripheral blood stem cell transplantation with reduced-intensity conditioning in acute myelogenous leukemia and myelodysplastic syndromes

Once-daily intravenous busulfan and fludarabine: clinical and pharmacokinetic results of a myeloablative, reducedtoxicity conditioning regimen for allogeneic stem cell transplantation in AML and MDS

Conditioning with 8Gy total body irradiation and fludarabine for allogeneic hematopoietic stem cell transplantation in acute myeloid leukemia

Definition of refractoriness against conventional chemotherapy in acute myeloid leukemia: a proposal based on the results of retreatment by thioguanine, cytosine arabinoside, and daunorubicin (TAD 9) in 150 patients with relapse after standardized first line therapy

Multivariate analysis of prognostic factors in patients with refractory and relapsed acute myeloid leukemia undergoing sequential high-dose cytosine arabinoside and mitoxantrone (S-HAM) salvage therapy: relevance of cytogenetic abnormalities

Improved results in primary refractory AML by sequential treatment with chemotherapy, reduced intensity conditioning for allogeneic stem cell transplantation and prophylactic DLT

Double induction strategy for acute myeloid leukemia: the effect of high-dose cytarabine with mitoxantrone instead of standard-dose cytarabine with daunorubicin and 6-thioguanine: a randomized trial by the German AML Cooperative Group

Prognostic factors for selecting curative therapy for adult acute myeloid leukemia

Pretreatment cytogenetic abnormalities are predictive of induction success, cumulative incidence of relapse, and overall survival in adult patients with de novo acute myeloid leukemia: results from Cancer and Leukemia Group B (CALGB 8461)

on behalf of the Medical Research Council Addult and Children's Leukemia Working Parties (1998) The importance of diagnostic cytogenetics on outcome in AML: Analysis of 1,612 patients entered into the MRC AML 10 trial

on behalf of the Medical Research Council Addult and Children's Leukemia Working Parties (2001) The predictive value of hierarchical cytogenetic classification in older adults with acute myeloid leukemia (AML): analysis of 1065 patients entered into the United Kingdom Medical Research Council AML 11 trial

Morphologic dysplasia in de novo acute myeloid leukemia (AML) is related to unfavorable cytogenetics but has no independent prognostic relevance under the conditions of intensive induction therapy: results of a multiparameter analysis from the German AML Cooperative Group studies

Double induction strategy for acute myeloid leukemia: The effect of high-dose cytarabine with mitoxantrone instead of standard-dose cytarabine with daunorubicin and 6-thioguanine: A randomized trial by the German AML Cooperative Group

Patients with de novo acute myeloid leukaemia and complex karyotype aberrations show a poor prognosis despite intensive treatment: A study of 90 patients

AML with 11q23/MLL abnormalities as defined by the WHO classification: incidence, partner chromosomes, FAB subtype, age distribution, and prognostic impact in an unselected series of 1897 cytogenetically analyzed AML cases

Karyotype is an independent prognostic parameter in therapy-related acute myeloid leukemia (t-AML): an analysis of 93 patients with t-AML in comparison to 1091 patients with de novo AML

Cytogenetics in acute myeloid leukemia

A new prognostic score for patients with acute myeloid leukemia (AML): Five prognostic subgroups can clearly be separated based on cytogenetics and early blast clearance in the 1992 and 1999 trials of the German AML Cooperative Group including 1001 patients

Early blast clearance by remission induction therapy is a major independent prognostic factor for both achievement of complete remission and long-term outcome in acute myeloid leukemia: data from the German AML Cooperative Group (AMLCG)

Underrepresentation of patients 65 years of age or older in cancer-treatment trials

Karyotype and age in acute myeloid leukemia. Are they linked?

Dependence of age-specific incidence of acute myeloid leukemia on karyotype

Population-based age-specific incidences of cytogenetic subgroups of acute myeloid leukemia

The influence of age on prognosis of de novo acute myeloid leukemia differs according to cytogenetic subgroups

Cytoplasmic nucleophosmin in acute myelogenous leukemia with a normal karyotype

Nucleophosmin gene mutations are predictors of favorable prognosis in acute myelogenous leukemia with a normal karyotype

Role of nucleophosmin in embryonic development and tumorigenesis

The protein tyrosine kinase inhibitor SU5614 inhibits FLT3 and induces growth arrest and apoptosis in AML-derived cell lines expressing a constitutively activated FLT3

The AML1-ETO fusion gene and the FLT3 length mutation collaborate in inducing acute leukemia in mice

CEBPA mutations in younger adults with acute myeloid leukemia and normal cytogenetics: prognostic relevance and analysis of cooperating mutations

AML1-ETO downregulates the granulocytic differentiation factor C/EBPalpha in t(8;21) myeloid leukemia

Use of gene-expression profiling to identify prognostic subclasses in adult acute myeloid leukemia

Prognostically useful gene-expression profiles in acute myeloid leukemia

Genetics of myeloid malignancies: pathogenetic and clinical implications

Clinical and biologic activity of the farnesyltransferase inhibitor R115777 in adults with refractory and relapsed acute leukemias: a phase 1 clinical-laboratory correlative trial

The roles of FLT3 in hematopoiesis and leukemia

Inhibition of mutant FLT3 receptors in leukemia cells by the small molecule tyrosine kinase inhibitor PKC412

FLT3 internal tandem duplication mutations associated with human acute myeloid leukemias induce myeloproliferative disease in a murine bone marrow transplant model

PML/RARalpha and FLT3-ITD induce an APL-like disease in a mouse model

The presence of a FLT3 internal tandem duplication in patients with acute myeloid leukemia (AML) adds important prognostic information to cytogenetic risk group and response to the first cycle of chemotherapy: analysis of 854 patients from the United Kingdom Medical Research Council AML 10 and 12 trials

Prognostic implication of FLT3 and N-RAS gene mutations in acute myeloid leukemia

Impact of FLT3 mutations and promyelocytic leukaemia-breakpoint on clinical characteristics and prognosis in acute promyelocytic leukaemia

Prognostic significance of FLT3 internal tandem duplication and tyrosine kinase domain mutations for acute myeloid leukemia: a meta-analysis

Absence of the wild-type allele predicts poor prognosis in adult de novo acute myeloid leukemia with normal cytogenetics and the internal tandem duplication of FLT3: a cancer and leukemia group B study

Suppression of leukemia expressing wild-type or ITD-mutant FLT3 receptor by a fully human anti-FLT3 neutralizing antibody

Selective apoptosis of tandemly duplicated FLT3-transformed leukemia cells by Hsp90 inhibitors

The protein tyrosine kinase inhibitor SU5614 inhibits FLT3 and induces growth arrest and apoptosis in AML-derived cell lines expressing a constitutively activated FLT3

CT53518, a novel selective FLT3 antagonist for the treatment of acute myelogenous leukemia (AML)

Inhibition of KIT tyrosine kinase activity: a novel molecular approach to the treatment of KIT-positive malignancies

SU11248 is a novel FLT3 tyrosine kinase inhibitor with potent activity in vitro and in vivo

SU5416, a small molecule tyrosine kinase receptor inhibitor, has biologic activity in patients with refractory acute myeloid leukemia or myelodysplastic syndromes

Single-agent CEP-701, a novel FLT3 inhibitor, shows biologic and clinical activity in patients with relapsed or refractory acute myeloid leukemia

Phase I and pharmacokinetic study of PKC412, an inhibitor of protein kinase C

Patients with acute myeloid leukemia and an activating mutation in FLT3 respond to a small-molecule FLT3 tyrosine kinase inhibitor

A Randomized Phase II Trial of the Tyrosine Kinase Inhibitor PKC412 in Patients (pts) with Acute Myeloid Leukemia (AML)/ High-Risk Myelodysplastic Syndrome (MDS) Characterized by Wild-Type (WT) or Mutated FLT3

Phase I clinical results with MLN518, a novel FLT3 antagonist: tolerability, pharmacokinetics, and pharmacodynamics

Phase II Evaluation of the Tyrosine Kinase Inhibitor MLN518 in Patients with Acute Myeloid Leukemia (AML) Bearing a FLT3 Internal Tandem Duplication (ITD) Mutation

In vitro studies of a FLT3 inhibitor combined with chemotherapy: sequence of administration is important to achieve synergistic cytotoxic effects

Open-Label Study of Lestaurtinib (CEP-701), an Oral FLT3 Inhibitor, Administered in Sequence with Chemotherapy in Patients with Relapsed AML Harboring FLT3 Activating Mutations: Clinical Response Correlates with Successful FLT3 Inhibition

Phase I Study of PKC412 and Oral FLT3 Kinase Inhibitor in Sequential and Concomitant Combinations wiht Daunorubicin and Cytarabine (DAA) Induction and High-Dose Cytarabine (HDAra-C) Consolidation in Newly Diagnosed Patients with AML

Phase IB Study of PKC412, an Oral FLT3 Kinase Inhibitor, in Sequential and Simultaneous Combinations with Daunorubicin and Cytarabine (DA) Induction and High-Dose Cytarabine Consolidation in Newly Diagnosed Patients with AML

Molecular remission in PML/RAR alpha-positive acute promyelocytic leukemia by combined all-trans retinoic acid and idarubicin (AIDA) therapy. Gruppo Italiano-Malattie Ematologiche Maligne dell'Adulto and Associazione Italiana di Ematologia ed Oncologia Pediatrica Cooperative Groups

A randomized comparison of all transretinoic acid (ATRA) followed by chemotherapy and ATRA plus chemotherapy and the role of maintenance therapy in newly diagnosed acute promyelocytic leukemia. The European APL Group

All-trans retinoic acid in acute promyelocytic leukemia: long-term outcome and prognostic factor analysis from the North American Intergroup protocol

Riskadapted treatment of acute promyelocytic leukemia with all-trans-retinoic acid and anthracycline monochemotherapy: a multicenter study by the PETHEMA group

Relapse of acute promyelocytic leukemia after 10 years long-term remission

Late relapse of acute promyelocytic leukemia treated with all-trans retinoic acid and chemotherapy: report of two cases

Late relapses in APL treated with ATRA and anthracycline based chemotherapy: the European APL Group experience (APL91 and APL 93 trials)

Relapse in the external auditory canal of acute promyelocytic leukemia after treatment with alltrans retinoic acid

Extramedullary involvement in patients with acute promyelocytic leukemia: a report of seven cases

Extramedullary relapse after all-trans retinoic acid treatment in acute promyelocytic leukemia-the occurrence of retinoic acid syndrome is a risk factor

Ear involvement in acute promyelocytic leukemia at relapse: a disease-associated 'sanctuary'?

Extramedullary relapse in a patient with acute promyelocytic leukemia: successful treatment with arsenic trioxide, all-trans retinoic acid and gemtuzumab ozogamicin therapies

Extramedullary involvement at relapse in acute promyelocytic leukemia patients treated or not with all-trans retinoic acid: a report by the Gruppo Italiano Malattie Ematologiche dell'Adulto

Randomized study of CODE versus alternating CAV/EP for extensive-stage small-cell lung cancer: an Intergroup Study of the National Cancer Institute of Canada Clinical Trials Group and the Southwest Oncology Group

Clinical features and outcome of patients with acute promyelofytic leukemia presenting CD56 antigen expression related with the PETHEMA LPA99 trial

Independent prognostic significance of FLT3 internal tandem duplication (ITD) mutations in acute promyelocytic leukemia treatment, a ECOG protocol

Alterations of the FLT3 gene in acute promyelocytic leukemia: association with diagnostic characteristics and analysis of clinical outcome in patients treated with the Italian AIDA protocol

Impact of FLT3 mutations and promyelocytic leukaemia-breakpoint on clinical characteristics and prognosis in acute promyelocytic leukaemia

Prognostic implication of FLT3 and Ras gene mutations in patients with acute promyelocytic leukemia (APL): a retrospective study from the European APL Group

Relationship between FLT3 mutation status, biologic characteristics, and response to targeted therapy in acute promyelocytic leukemia

AIDA-The Italian way of treating acute promyelocytic leukemia (APL). The final act

The recent JALSG study for newly diagmnosed patients with acute promyelocytic leukemia (APL)

Postremission therapy in adult acute myeloid leukemia (AML): a randomized comparison of intensified consolidation therapy without amintenance against convenmtional consolidation with maintenance therapy-JALSG AML-97 trial

Autologous bone marrow transplantation for acute promyelocytic leukemia in second remission: prognostic relevance of pretransplant minimal residual disease assessment by reverse-transcription polymerase chain reaction of the PML/RAR alpha fusion gene

Stem cell transplantation for acute promyelocytic leukemia in the ATRA era: a survey of the European Blood and Marrow TRansplantation Group (EBMT)[abstr]

BAVC regimen and autologous bone marrow transplantation for APL patients in second molecular remission: updated results

Autologous and allogeneic stem-cell transplantation as salvage treatment of acute promyelocytic leukemia initially treated with all-trans retinoic acid: a retrospective analysis of the European acute promyelocytic leukemia group

Allogeneic stem cell transplantation for advanced acute promyelocytic leukemia: results in patients treated in second molecular remission or with molecularly persistent disease

Treatment of newly diagnosed acute promyelocytic leukemia (APL): the impact of high-dose Ara-C

Front-line treatment of acute promyelocytic leukemia with AIDA induction followed by risk-adapted consolidation: results of the AIDA-2000 trial of the Italian GIMEMA Group

Sequential targeted therapy for relapsed acute promyelocytic leukemia with alltrans retinoic acid and anti-CD33 monoclonal antibody M195

Molecular remission induction with retinoic acid and anti-CD33 monoclonal antibody HuM195 in acute promyelocytic leukemia

Gemtuzumab ozogamicin (Mylotarg) as a single agent for molecularly relapsed acute promyelocytic leukemia

Prolonged molecular remission in advanced acute promyelocytic leukaemia after treatment with gemtuzumab ozogamicin (Mylotarg CMA-676)

A single administration of gemtuzumab ozogamicin for molecular relapse of acute promyelocytic leukemia

Efficacy of gemtuzumab ozogamicin on ATRA-and arsenic-resistant acute promyelocytic leukemia (APL) cells

Arsenic trioxide: new clinical experience with an old medication in hematologic malignancies

United States multicenter study of arsenic trioxide in relapsed acute promyelocytic leukemia

Arsenic trioxide therapy in relapsed or refractory Japanese patients with acute promyelocytic leukemia: updated outcomes of the phase II study and postremission therapies

All-trans retinoic acid/As203 combination yields a high quality remission and survival in newly diagnosed acute promyelocytic leukemia

Use of all-trans retinoic aide (ATRA) + arsenic trioxide (ATO) to eliminate or minimize use of chemotherapy (CT) in untreated acute promyelotcytci leukemia (APL)

An efficient therapeutic approach to patients with acute promyelocytic leukemia using a combination of arsenic trioxide with low-dose all-trans retinoic acid

Durable remissions with single agent As203 in the treatment of newly diagnosed cases of acute promyelocytic leukemia: risk stratification within this group and potential impact on future algorithms

Molecular remission with arsenic trioxide in patients with newly diagnosed acute promyelocytic leukemia

Internal tandem duplication and Asp835 mutations of the FMS-like tyrosine kinase 3 (FLT3) gene in acute promyelocytic leukemia

FLT3-activating mutations in acute promyelocytic leukaemia: a rationale for risk-adapted therapy with FLT3 inhibitors

All-trans-retinoic acid induces CD52 expression in acute promyelocytic leukemia

Vascular endothliae growth factor (VEGF) is a critical autocrine survival factor in acute promyelocytic leukemia (APL)

Angiogenesis in acute promyelocytic leukemia: induction by vascular endothelial growth factor and inhibition by all-trans retinoic acid

Anti-leukemic and anti-angiogenesis efficacy of arsenic trioxide in new cases of acute promyelocytic leukemia

Risk Adapted Stem Cell Transplantation?

Autologous or allogeneic bone marrow transplantation compared with intensive chemotherapy in acute myelogenous leukemia

The influence of HLA-matched sibling donor availability on treatment outcome for patients with AML: an analysis of the AML 8A study of the EORTC Leukemia Cooperative Group and GIMEMA

The value of allogeneic bone marrow transplantation in patients with acute myeloid leukaemia at differing risk of relapse: results of the UK MRC AML 10 trial

Current controversie: which patients with acute myeloid leulaemia should receive a bone marrow transplantation? -An adult treater's view

Allogeneic compared to autologous stem cell transplantation in the treatment of patients < 46 years old with acute myeloid leukemia (AML) in first complete remission (CR1): an intention to treat analysis of the EORTC/GIMEMA AML-10 trial

Role of allogeneic stem cell transplantation in current treatment of acute myeloid leukemia

Molecular Heterogeneity and Its Prognostic Significance in Acute Myeloid Leukemia (AML) With Normal Cytogenetics

BAALC expression predicts clinical outcome of de novo acute myeloid leukemia patients with normal cytogenetics: a Cancer and Leukemia Group B study

Acute myeloid leukemia with complex karyotypes and abnormal chromosome 21: amplification discloses overexpression of APP, ETS2, and ERG genes

BAALC expression and FLT3 internal tandem duplication mutations in acute myeloid leukemia patients with normal cytogenetics: prognostic implications

BAALC expression and FLT3 internal tandem duplication mutations in acute myeloid leukemia patients with normal cytogenetics: prognostic implications

Risk assessment in patients with acute myeloid leukemia and a normal karyotype

Curative impact of intensification with high-dose cytarabine (HiDAC) in acute myeloid leukemia (AML) varies by cytogenetic group

Frequency of prolonged remission duration after high-dose cytarabine intensification in acute myeloid leukemia varies by cytogenetic subtype

) Prevalence, clinical profile, and prognosis of NPM mutations in AML with normal karyotype

Use of gene-expression profiling to identify prognostic subclasses in adult acute myeloid leukemia

Pretreatment cytogenetic abnormalities are predictive of induction success, cumulative incidence of relapse, and overall survival in adult patients with de novo acute myeloid leukemia: results from Cancer and Leukemia Group B (CALGB 8461)

Rearrangement of ALL1 (MLL) in acute myeloid leukemia with normal cytogenetics

Revised recommendations of the International Working Group for Diagnosis, Standardization of Response Criteria, Treatment Outcomes, and Reporting Standards for Therapeutic Trials in Acute Myeloid Leukemia

Prognostic significance of partial tandem duplications of the MLL gene in adult patients 16 to 60 years old with acute myeloid leukemia and normal cytogenetics: a study of the Acute Myeloid Leukemia Study Group Ulm

Mutant nucleophosmin (NPM1) predicts favorable prognosis in younger adults with acute myeloid leukemia and normal cytogenetics: interaction with other gene mutations

Cytoplasmic nucleophosmin in acute myelogenous leukemia with a normal karyotype

Outcome of induction and postremission therapy in younger adults with acute myeloid leukemia with normal karyotype: a Cancer and Leukemia Group B study

Prognostic significance of activating FLT3 mutations in younger adults (16 to 60 years) with acute myeloid leukemia and normal cytogenetics: a study of the AML Study Group Ulm

CEBPA mutations in younger adults with acute myeloid leukemia and normal cytogenetics: prognostic relevance and analysis of cooperating mutations

Molecular classification of cancer: class discovery and class prediction by gene expression monitoring

The importance of diagnostic cytogenetics on outcome in AML: analysis of 1,612 patients entered into the MRC AML 10 trial

World Health Organization classification of tumours. Pathology and genetics of tumours of haematopoietic and lymphoid tissues

Variable prognostic value of FLT3 internal tandem duplications in patients with de novo AML and a normal karyotype

Monitoring of minimal residual disease in acute myeloid leukemia

Dose escalation studies of cytarabine, daunorubicin, and etoposide with and without multidrug resistance modulation with PSC-833 in untreated adults with acute myeloid leukemia younger than 60 years: final induction results of Cancer and Leukemia Group B study 9621

Detailed analysis of FLT3 expression levels in acute myeloid leukemia

FLT3 tyrosine kinase inhibitors

Abnormal cytogenetics at date of morphologic complete remission predicts short overall and disease-free survival, and higher relapse rate in adult acute myeloid leukemia: results from Cancer And Leukemia Group B study 8461

Molecular heterogeneity and prognostic biomarkers in adults with acute myeloid leukemia and normal cytogenetics

Overexpression of the ETS-related gene, ERG, predicts a worse outcome in acute myeloid leukemia with normal karyotype: a Cancer and Leukemia Group B study

Independent validation of prognostic relevance of a previously reported gene-expression signature in acute myeloid leukemia (AML) with normal cytogenetics (NC): a Cancer and Leukemia Group B (CALGB) study. Blood 106:223a 31. Mró zek K, Heerema NA

Acute myeloid leukemia prognostic factors: from cytogenetics to chip

ETS transcription factors: possible targets for cancer therapy

Biologic and clinical significance of the FLT3 transcript level in acute myeloid leukemia

Favorable prognostic significance of CEBPA mutations in patients with de novo acute myeloid leukemia: a study from the Acute Leukemia French Association (ALFA)

Practice points, consensus, and controversial issues in the management of patients with newly diagnosed acute promyelocytic leukemia

Nucleophosmin gene mutations are predictors of favorable prognosis in acute myelogenous leukemia with a normal karyotype

Mutation of CEBPA in familial acute myeloid leukemia

Patients with acute myeloid leukemia and an activating mutation in FLT3 respond to a small-molecule FLT3 tyrosine kinase inhibitor

Analysis of FLT3-activating mutations in 979 patients with acute myelogenous leukemia: association with FAB subtypes and identification of subgroups with poor prognosis

Prognostically useful gene-expression profiles in acute myeloid leukemia

Absence of the wild-type allele predicts poor prognosis in adult de novo acute myeloid leukemia with normal cytogenetics and the internal tandem duplication of FLT3: a Cancer and Leukemia Group B study

The MLL partial tandem duplication: evidence for recessive gain-of-function in acute myeloid leukemia identifies a novel patient subgroup for molecular-targeted therapy

Germany Abstract After rapid improvement of treatment results in adult acute lymphoblastic leukemia (ALL) from less than 10% to 35-40% in the past decades, more recently progress was only observed within subtype directed treatment approaches. In addition a borderline for further intensification of chemotherapy appears to be reached in adult ALL patients. New, preferable non-chemotherapy, approaches are therefore urgently required. One of those is targeted therapy with monoclonal antibodies (MoAbs)

New approaches in acute lymphoblastic leukemia in adults: Where do we go?

New treatment options in adult ALL

Monoclonal antibody therapies in leukemias

Treatment with monoclonal antibodies in acute lymphoblastic leukemia: current knowledge and future prospects

Rituximab: mechanism of action and resistance

CHOP chemotherapy plus rituximab compared with CHOP alone in elderly patients with diffuse large-B-cell lymphoma

Clinical use of rituximab in haematological malignancies

Improved outcome in adult B-cell acute lymphoblastic leukemia

Short Intensive Chemotherapy with Rituximab Seems Successful in Burkitt NHL, Mature B-ALL and Other High-Grade B-NHL

Successful treatment of Burkitt's NHL and other high-grade NHL according to a protocol for mature B-ALL

Subtype adjusted therapy improves outcome of elderly patients with acute lymphoblastic leukemia

Chemo-Immunotherapy with Hyper-CVAD Plus Ritixumab for Adult Burkitt's and Burkitt's Type Lymphoma (BL) or Acute Lymphoblastic Leukemia (B-ALL)

Results of a shortened, dose reduced treatment protocol in elderly patients with acute lymphoblastic leukemia (ALL)

Update of the Modified Hyper-CVAD Regimen with or without Rituximab in Newly Diagnosed Adult Acute Lymphocytic Leukemia (ALL)

Rituximab with interleukin-2 after autologous bone marrow transplantation for acute lymphocytic leukemia in second remission

Rituximab can be useful as treatment for minimal residual disease in bcr-abl-positive acute lymphoblastic leukemia

Rituximab in three children with relapsed/refractory B-cell acute lymphoblastic leukaemia/Burkitt non-Hodgkin's lymphoma

Induction of long-term remission of a relapsed childhood B-acute lymphoblastic leukemia with rituximab chimeric anti-CD20 monoclonal antibody and autologous stem cell transplantation

Chronic myeloid leukemia -Advances in biology and new approaches to treatment

In vitro studies of the combination of imatinib mesylate (Gleevec) and arsenic trioxide (Trisenox) in chronic myelogenous leukemia

Chronic myeloid leukemia and interferon-alpha: a study of complete cytogenetic responders

Effects of a selective inhibitor of the Abl tyrosine kinase on the growth of Bcr-Abl positive cells

Response and resistance in 300 patients with BCR-ABL positive leukemias treated with imatinib in a single center

The IRIS study: International randomized study of interferon and low-dose ara-C versus STI571 (imatinib) in patients with newly-diagnosed chronic phase chronic myeloid leukemia

How long will chronic phase CML pateints treated with imatinib live?

Accurate and rapid analysis of residual disease in patients with CML using specific fluorescent hybridization probes for real time quantitative RT-PCR

Dynamics of BCR-ABL mRNA expression in first line therapy of chronic myelogenous leukemia patients with imatinib or interferon /ara-C

Frequency of major molecular responses to imatinib or interferon alfa plus cytarabine in newly diagnosed chronic myeloid leukemia

Imatinib therapy in chronic myelogenous leukemia: strategies to avoid and overcome resistance

Molecular and chromosomal mechanisms of resistance to imatinib (STI571) therapy

Detection of BCR-ABL mutations in patients with CML treated with imatinib is virtually always accompanied by clinical resistance, and mutations in the ATP phosphate-binding loop (P-loop) are associated with a poor prognosis

High-dose imatinib mesylate therapy in newly diagnosed Philadelphia chromosome-positive chronic phase chronic myeloid leukemia

Imatinib and pegylated human recombinant interferon-2b in early chronic phase chronic myeloid leukemia

Imatinib And Beyond -The New CML-Study IV. A Randomised Controlled Comparison of Imatinib vs. Imatinib/Interferon-Alpha vs. Imatinib/Low-Dose AraC vs. Interferon-Alpha Standard Therapy in Newly Diagnosed Chronic Phase Chronic Myeloid Leukemia

Interferon-alpha, but not the ABL-kinase inhibitor imatinib (STI571), induces expression of myeloblastin and a specific T-cell response in chronic myeloid leukemia

Characterization of AMN107, a selective inhibitor of native and mutant Bcr-Abl

Overriding imatinib resistance with a novel ABL kinase inhibitor

Risk assessment for patients with chronic myeloid leukemia before allogeneic blood or marrow transplantation

Forty percent (111/277) of patients had 5% leukemic blast cells in bone marrow aspirates taken at 5 to 11 days after the first dose of GO. These 111 patients included 56 of 66 (85%) OR patients who had marrow analysis performed. Ten of 66 OR patients (15%) had >5% blast cells in bone marrow aspirates after the first dose of GO but subsequently achieved remission by the end of treatment. Relapsefree survival (RFS) was similar in both CR and CRp patients (P=0.72). The median RFS was 6.4 months for patients with CR, 4.5 months for patients with CRp, and 5.2 months for the combined group (CR+CRp). A significant difference in RFS was observed between patients <60 years and patients 60 years (P=0.008). This difference may have been influenced by postremission treatment options, especially hematolopoietic cell transplantation (HCT). with CR, 12.9 months for patients with CRp, and 4.2 months for patients who survived the treatment period but did not achieve remission (CR or CRp vs NR, P<0.001). Median overall survival for CR patients <60 years was 17.2 months and for CRp patients was 18.4 months. For CR or CRp patients 60 years, median overall survivals were 11.7 or 11.4 months, respectively. Although expected incidences of grade 3 or 4 neutropenia (98%) and thrombocytopenia (99%) were observed, grade 3 or 4 sepsis (17%) and pneumonia (8%) was relatively infrequent

Synergy of demethylation and histone deacetylase inhibition in the reexpression of genes silenced in cancer

Demethylation of a hypermethylated p15/INK4B gene in patients with myelodysplastic syndrome by 5-Aza-2'-deoxycytidine treatment

Epigenetics in human disease and prospects for epigenetic therapy

Azacitidine: 10 years later

Changes in promoter methylation and gene expression in patients with MDS and MDS-AML treated with 5-azacytidine and sodium phenylbutyrate

Phase 1 study of lowdose prolonged exposure schedules of the hypomethylating agent 5-aza-2'-deoxycytidine (decitabine) in hematopoietic malignancies

Cellular differentiation, cytidine analogs and DNA methylation

Nienhuis AW (1982) 5-Azacytidine selectively increases yglobin synthesis in a patient with + thalassemia

Low-dose 5-azacytidine is ineffective for remission induction in patients with acute myeloid leukemia

Continued Low-Dose Decitabine (DAC) is an active first line treatment of older AML patients: First results of a multicenter phase II study

Developmental regulation of myeloid gene expression and demethylation during ex vivo culture of peripheral blood progenitor cells

DNA methyltransferase inhibitors and the development of epigenetic cancer therapies

Azacitidine (Vidaza ) treatment response assessed using three alternative dosing schedules in patients with myelodysplastic syndromes (MDS)

Di Fiore PP (1984) 5-Aza-2'-deoxycytidine induces terminal differentiation of leukemic blasts from patients with acute myeloid leukemias

5-Aza-2'-deoxycytidine as a differentiation inducer in acute myeloid leukaemias and myelodysplastic syndromes of the elderly

Clinical studies with fetal hemoglobinenhancing agents in sickle cell disease

Decitabine (5-aza-2'-deoxycytidine; decitabine) plus daunorubicin as a first line treatment in patients with acute myeloid leukemia: preliminary observations

Randomized controlled trial of azacitidine in patients with the myelodysplastic syndrome: a study of the cancer and leukemia group B

An epigenetic approach to the treatment of advanced MDS; the experience with the DNA demethylation agent 5-aza-2´-deoxycytidine (decitabine) in 177 patients

A. Low-dose 5-aza-2'-deoxycytidine, a DNA hypomethylating agent, for the treatment of high-risk myelodysplastic syndrome: a multicenter phase II study in elderly patients

A randomized phase-II study on the effects of 5-aza-2'-deoxycytidine combined with either amsacrine or idarubicin in patients with relapsed acute leukemia: an EORTC Leukemia Cooperative Group phase-II study

Gene Expression Profiles and Treatment Response in Acute References

Acute lymphoblastic leukaemia

Molecular pharmacodynamics in childhood leukemia

Gene-expression patterns in drug-resistant acute lymphoblastic leukemia cells and response to treatment

The expression of 70 apoptosis genes in relation to lineage, genetic subtype, cellular drug resistance, and outcome in childhood acute lymphoblastic leukemia

Asparagine synthetase expression is linked with L-asparaginase resistance in TEL-AML1 negative, but not in TEL-AML1 positive pediatric acute lymphoblastic leukemia

Discovery of novel molecular classification schemes and genes predictive of outcome in leukemia

Expression of the Outcome Predictor in Acute Luekmeia 1 (OPAL1) gene is not related to outcome in patients treated on contemporary COALL or

Rudin CM, den Boer ML, Pieters R Sensitizing effect of glycolysis inhibition on prednisolone resistance in acute lymphoblastic leukemia

We would like to express our sincere thanks to all the hospitals and investigators in Germany, Austria, Switzerland and the Czech Republic who participated in study AML-BFM 98 for their valuable assistance.

We are indebted to all physicians and staff at the participating centers. Imatinib used in this study was kindly provided by Novartis Pharmaceuticals (Basel, Switzerland). 

Current standard treatment strategy for patients with acute myeloid leukemia (AML) younger than 60 years consists of one or two induction courses with cytosine arabinoside and an anthracycline followed in the case of a complete remission by one or more intensive consolidation (IC) chemotherapy courses.There is no consensus about the subsequent treatment modality. Some prefer only chemotherapy i.e. continuation of consolidation courses and/or maintenance chemotherapy; others proceed with some form of autologous (auto-SCT) or allogeneic (allo-SCT) stem cell transplantation. Several large studies comparing IC courses with stem cell transplantation report contradictory results. In the AML-8A trial of the EORTC and GIMEMA Leukemia Groups, it has been shown that auto-SCT leads to a significant longer DFS than a second intensive consolidation course (48% vs 30% at 4 years)1. In this trial, in patients younger than 46 years, an 8% survival superiority (48% v 40%) at 6 years for the donor stem cell transplantation group (containing all patients with a HLAidentical family donor) compared to the no donor group (those who were eligible for an auto-SCT or IC) has been observed as well2. This difference was not significant (P=0.24) due, in part, to an insufficient numbers of patients per treatment group. Similarly Burnett et al reported in their recent MRC AML-10 trial in which patients after four chemotherapy courses continued with allo-or auto-SCT or no further treatment that the patients with a donor had a higher 7year DFS rate than the patients without a donor (50% versus 42%) with the most pronounced effect in the cytogenetically intermediate risk group3-4.The EORTC and GIMEMA investigators subsequently conducted a randomized study (AML-10) for patients under the age of 61 years consisting of one or two three-drug induction courses followed by an intensive consolidation course and, for those under the age of 46 years, an allogeneic stem cell transplantation in the case of an available family donor or an auto-SCT in the other patients5. Between November 1993 and December 1999, of 1198 patients aged < 46 years, 822 achieved CR. The study group constituted 734 patients who received IC: 293 had a sibling donor and 441 did not. Allo-SCT and auto-SCT was performed in 68.9% and 55.8%, respectively. Cytogenetics was successfully performed in 446 patients. Risk groups were: good (t(8;21), inv (16)), intermediate (NN or -Y only), bad/very bad (all others). Median follow-up was 4 years; 289 pts relapsed, 66 died in CR1, and 293 died. Intention-to-treat analysis revealed that the 4-year disease-free survival (DFS) rate of patients with a donor versus of those without a donor was 52.2% versus 42.2%, p=0.044; hazard ratio=0.80, 95% confidence interval (0.64 , 0.995), the relapse incidence was 30.4% versus 52.5%, death in CR1 was 17.4% versus 5.3%, and the survival rate was 58.3% vs 50.8% (p=0.18). The DFS rates in patients with and without a sibling donor were similar in patients with good/intermediate risk, but were 43.4% and 18.4%, respectively, in patients with bad/very bad risk cytogenetics. In younger patients (15-35 yrs) , the difference was more pronounced.Recently a compilation of the results of several consecutive HOVON-SAKK studies showed a DFS and survival advantage for patients in complete remission with a donor vs those without a donor6.. This was true for patients with intermediate as well as bad risk cytogenetics.None of the mentioned randomized trials did show an advantage of an allo-SCT for patients with good risk cytogenetics.Currently the EORTC and GIMEMA Leukemia Groups assess in their AML-12 randomized phase III trial the efficacy and toxicity of HD-AraC (3 g/m2 q 12 hrs for 4 days) in combination with daunorubicin (50 mg/m2 for 3 days) and etoposide (50 mg/m2 for 5 days) vs SD-AraC (100 mg/m2 for 10 days) combined with the same drugs. All patients (pts) who reach a complete remission (CR) will receive one consolidation course consisting of ID-AraC (500 mg/m2 q 12 hrs for 6 days) and daunorubicin. Subsequently an allogeneic (allo-SCT) or an autologous stem cell transplantation (auto-SCT) is planned according to donor availability and age. A second randomization is performed after consolidation in patients without a donor: auto-SCT followed or not by maintenance with low dose IL-2 (4-8 x 106 IU s.c. for 5 days per month) during one year. From 1999 till October 2005, 1434 AML pts (APL excluded), age<61 years, from 65 centers (23 EORTC-LG and 42 GIMEMA) entered the trial. Currently 1330 pts have been randomized for induction and 372 pts postconsolidation. During the induction course toxicity profiles were similar in the 2 arms; however in the HD-AraC group the incidence of grade 3-4 liver transaminase abnormalities (15% vs 11.5%) and conjunctivitis (6% vs 0%) was higher. HD-AraC in the induction cycle had no impact on the organ toxicity during the consolidation course, but the platelet recovery (>50x109/l) was significantly longer (median 4.4 vs 3.1 weeks). The IL-2 schedule was well tolerated in most pts with fatigue (20%), rigor/chills (6.5%), arthralgia/myalgia (4%) as the main grade 3-4 toxicities. Among 628 pts randomized until December 2004 by EORTC centers and 6 large GIMEMA centers, with a median follow-up of 2.2 years, 481 reached CR. Among 443 pts who received a consolidation course, 63 pts went off-study due to toxicity/relapse or no information is still available, and 380 pts were still CR after consolidation: 163 had no donor, 144 had a donor and 69 have not been HLA typed (too old, other reasons). In these 3 groups the present estimates of the transplantation rates were: 59% (auto-SCT), 74% (allo-SCT) and 66% (auto-SCT), respectively. The 2-yr DFS rates (SE%) were 51% (5%), 65% (5%), and 55% (7%), respectively in the donor, no donor and not HLA typed group. Patients with a donor (N=144) had a longer DFS rate than patients without a donor (N=163): the estimated hazard ratio adjusted for age was 0.60, 95% CI (0.37, 0.97).

1. allo-SCT is currently the best available antileukemic treatment modality 2. cytogenetically good risk patients in first complete remission should not be considered candidates for allo-SCT (unless the transplantation related mortality decreases) 3. allo-SCT in AML in first complete remission in younger patients (15-35 yrs old) leads to a DFS and (possibly) survival advantage in patients with leukemia with intermediate or bad risk cytogenetics